Abuse-resistant amphetamine prodrugs

ABSTRACT

The invention describes compounds, compositions, and methods of using the same comprising a chemical moiety covalently attached to amphetamine. These compounds and compositions are useful for reducing or preventing abuse and overdose of amphetamine. These compounds and compositions find particular use in providing an abuse-resistant alternative treatment for certain disorders, such as attention deficit hyperactivity disorder (ADHD), ADD, narcolepsy, and obesity. Oral bioavailability of amphetamine is maintained at therapeutically useful doses. At higher doses bioavailability is substantially reduced, thereby providing a method of reducing oral abuse liability. Further, compounds and compositions of the invention decrease the bioavailability of amphetamine by parenteral routes, such as intravenous or intranasal administration, further limiting their abuse liability.

CROSS REFERENCE RELATED APPLICATIONS

This application is a continuation-in-part of U.S. application Ser. No. 11/400,304, filed Apr. 10, 2006, which in turn, (a) claims benefit under 35 U.S.C. 119(e) to U.S. Provisional Application Nos. 60/669,385 filed Apr. 8, 2005, 60/669,386 filed Apr. 8, 2005, 60/681,170 filed May 16, 2005, 60/756,548 filed Jan. 6, 2006, and 60/759,958 filed Jan. 19, 2006; (b) is a continuation-in-part of U.S. application Ser. No. 10/857,619 filed Jun. 1, 2004, which claims the benefit of under 35 U.S.C. 119(e) to U.S. Provisional Application Nos. 60/473,929 filed May 29, 2003 and 60/567,801 filed May 5, 2004; and (c) is a continuation-in-part of U.S. application Ser. No. 10/858,526 filed Jun. 1, 2004, which, in turn, is a continuation-in-part of international application PCT/US03/05525 filed Feb. 24, 2003, which claims priority to U.S. Provisional Application Nos. 60/358,368 filed Feb. 22, 2002 and 60/362,082 filed Mar. 7, 2002; application Ser. No. 10/858,526 also claims benefit under 35 U.S.C. 119(e) to U.S. Provisional Application Nos. 60/473,929 filed May 29, 2003 and 60/567,801 filed May 5, 2004. All of the above-identified applications are hereby incorporated by reference in their entirety.

FIELD OF THE INVENTION

The invention relates to amphetamine compounds, more particularly to amphetamine prodrugs comprising amphetamine covalently bound to a chemical moiety. The invention also relates to pharmaceutical compositions comprising the amphetamine compounds, and to methods of manufacturing, delivering, and using the amphetamine compounds. The invention further relates to a crystalline form of the amphetamine prodrug (l)-lysine-(d)-amphetamine dimesylate.

BACKGROUND OF THE INVENTION

Amphetamines stimulate the central nervous system (CNS) and have been used medicinally to treat various disorders including attention deficit hyperactivity disorder (ADHD), obesity, and narcolepsy. In children with ADHD, potent CNS stimulants have been used for several decades as a drug treatment given either alone or as an adjunct to behavioral therapy. While methylphenidate (Ritalin®) has been the most frequently prescribed stimulant, the prototype of the class, amphetamine (alpha-methyl phenethylamine) has been used all along and increasingly so in recent years. (Bradley C, Bowen M, “Amphetamine (benzedrine) therapy of children's behavior disorders.” American Journal of Orthopsychiatry 11: 92-103 (1941).

Because of their stimulating effects, amphetamines, including amphetamine derivatives and analogs, are subject to abuse. A user can become dependent over time on these drugs and their physical and psychological effects, even when the drugs are used for legitimate therapeutic purposes. Legitimate amphetamine users that develop drug tolerances are especially susceptible to becoming accidental addicts as they increase dosing in order to counteract their increased tolerance of the prescribed drugs. Additionally, it is possible for individuals to inappropriately self-administer higher than prescribed quantities of the drug or to alter either the product or the route of administration (e.g., inhalation (snorting), injection, and smoking), potentially resulting in immediate release of the active drug in quantities larger than prescribed. When taken at higher than prescribed doses, amphetamines can cause temporary feelings of exhilaration and increased energy and mental alertness.

Recent developments in the abuse of prescription drug products increasingly raise concerns about the abuse of amphetamine prescribed for ADHD. The National Survey on Drug Use and Health (NSDUH), estimates that in 2003, 1.2 million Americans aged 12 and older abused stimulants, such as amphetamines. The high abuse potential has earned amphetamines Schedule II status according to the Controlled Substances Act (CSA). Schedule II classification is reserved for those drugs that have accepted medical use but have the highest potential for abuse.

Extended release formulations of amphetamines, e.g., Adderall XR®, have an increased abuse liability relative to the single dose tablets because each tablet of the sustained release formulation contains a higher concentration of amphetamine. It may be possible for substance abusers to obtain a high dose of amphetamine with rapid onset by crushing the tablets into powder and snorting it or by dissolving the powder in water and injecting it. Sustained release formulations may also provide uneven release.

Additional information about amphetamines and amphetamine abuse can be found in U.S. Publication No. 2005/0054561 (U.S. Ser. No. 10/858,526).

The need exists for additional amphetamine compounds, especially abuse resistant amphetamine compounds. Further, the need exists for amphetamine pharmaceutical compositions that provide sustained release and sustained therapeutic effect.

SUMMARY OF THE INVENTION

The present invention provides amphetamine prodrugs, and salts thereof, comprising amphetamine covalently bound to a chemical moiety. A preferred amphetamine prodrug is (l)-lysine-(d)-amphetamine dimesylate (also known as lisdexamphetamine dimesylate). Lisdexamphetamine dimesylate can be in crystalline form, and can be anhydrous (i.e., crystalline anhydrous lisdexamphetamine dimesylate).

One embodiment of the invention is crystalline lisdexamphetamine dimesylate which exhibits an X-ray powder diffraction (XRPD) pattern having at least one peak in degrees 2Θ±0.2° 2Θ selected from 4.5, 9.0, 12.0, 15.7, and 16.3. In another embodiment, the crystalline lisdexamphetamine dimesylate exhibits 2, 3, 4, or all of the aforementioned peaks. The crystalline lisdexamphetamine dimesylate can exhibit an XRPD substantially as shown in FIG. 77.

Another embodiment of the invention is crystalline lisdexamphetamine dimesylate having a melting point onset as determined by differential scanning calorimetry (DSC) at about 194.7° C.

Yet another embodiment is crystalline lisdexamphetamine dimesylate that exhibits a single crystal X-ray crystallographic analysis at 150 K with crystal parameters that are approximately equal to the following:

Parameter Value Space group P2₁/a Cell Dimensions a (Å) 10.25 b (Å) 11.28 c (Å) 19.35 β (°) 94.12 Volume (Å³) 2232.1 Z (Molecules/unit cell) 4 Density (g/cm³) 1.359

Still, another embodiment is crystalline lisdexamphetamine dimesylate having at least one of a d₁₀ ranging from about 1 to about 10 μm; a d₅₀ ranging from about 8 to about 40 μm; and a d₉₀ particle size ranging from about 25 to about 90 μm.

The crystalline lisdexamphetamine dimesylate can be orally administered to treat attention deficit hyperactivity disorder as well as the other disorders described herein. For example, one embodiment is a method of treating attention deficit hyperactivity disorder in a patient (e.g., a child or adult) in need thereof by administering an effective amount of the crystalline lisdexamphetamine dimesylate.

In yet another aspect of the invention, a method for preparing crystalline lisdexamphetamine dimesylate is provided. The method entails reacting a di-amine protected l-lysine-d-amphetamine with methane sulfonic acid to produce lisdexamphetamine dimesylate.

Another embodiment of the invention is directed to a method of reducing patient to patient variability of amphetamine levels among a group of patients. The method entails daily (preferably once daily) oral administration to each patient in the group of a prodrug of amphetamine or a pharmaceutically acceptable salt of the prodrug, wherein the amphetamine is covalently bound to a peptide comprising 1 to 10 amino acids. In one preferred embodiment, a pharmaceutical composition consisting essentially of the prodrug of amphetamine or a pharmaceutically acceptable salt thereof is administered. A preferred prodrug is L-lysine-d-amphetamine and pharmaceutically acceptable salts thereof, such as L-lysine-d-amphetamine dimesylate.

Yet another embodiment of the invention is a method of treating obesity, cancer related fatigue, excessive daytime sleepiness in patients suffering from obstructive sleep apnea, or narcolepsy, in a patient in need thereof by administering an effective amount of the aforementioned amphetamine prodrug or a pharmaceutically acceptable salt thereof or a pharmaceutical composition containing it. According to one preferred embodiment, the prodrug or pharmaceutically acceptable salt thereof is orally administered once daily. A preferred prodrug is L-lysine-d-amphetamine, and a preferred salt of the prodrug is L-lysine-d-amphetamine dimesylate.

Yet another embodiment of the invention is a method of treating depression or a depressive disorder (such as major depressive disorder) in a patient in need thereof by administering an effective amount of (a) the aforementioned amphetamine prodrug or a pharmaceutically acceptable salt thereof or a pharmaceutical composition containing it and (b) an antidepressant other than mirtazapine.

Yet another embodiment is a method of treating ADHD in a patient in need thereof while minimizing the risk of adverse side effects due to the mode of treatment, comprising:

(a) marketing to doctors an oral pharmaceutical composition comprising L-lysine-d-amphetamine or a pharmaceutically acceptable salt thereof for the treatment of ADHD and as having a lower interpatient variation than an oral formulation containing d-amphetamine; (b) diagnosing a patient as suffering from ADHD by a doctor; (c) prescribing the oral pharmaceutical composition comprising L-lysine-d-amphetamine or a pharmaceutically acceptable salt thereof to the patient by the doctor in response to the marketing of the oral pharmaceutical composition and the diagnosis of the patient; and (d) orally administering the prescribed oral pharmaceutical composition to the patient. The term “marketing” refers to the act or process of selling a product, including, but not limited to, any offer for sale or sale of a product, including advertising The term “advertising” refers to notifying, informing, and/or apprising one or more individuals of information (e.g., the efficacy of a pharmaceutical product for treating or reducing an indication), such as by mass media, including, but not limited to, newspaper, magazine, and internet advertisements, television commercials, and billboard signs. The term “advertising” as used herein also includes including a statement that the pharmaceutical product can treat the indication(s) in the labeling (e.g., ADHD) for the pharmaceutical product.

Yet another embodiment is a method for treating a patient suffering from ADHD comprising the steps of:

(a) providing to the marketplace an oral pharmaceutical composition comprising L-lysine-d-amphetamine or a pharmaceutically acceptable salt thereof for the treatment of ADHD; (b) sensitizing a doctor to a product claim that the oral pharmaceutical composition has a lower interpatient variation than an oral formulation containing d-amphetamine; (c) diagnosing a patient as suffering from ADHD by the doctor; (d) selectively prescribing the oral pharmaceutical composition to the patient by the sensitized doctor in view of the diagnosis of the patient; and (e) orally administering the prescribed oral pharmaceutical composition to the patient.

Yet another embodiment is a method for treating a patient suffering from ADHD comprising the steps of:

(a) purchasing an oral pharmaceutical composition comprising L-lysine-d-amphetamine or a pharmaceutically acceptable salt thereof; (b) tendering a prescription for the patient suffering from ADHD before completing the purchase transaction, the prescription having been received in response to a diagnosis of ADHD by a doctor, the doctor (1) having been sensitized to a product claim that the oral pharmaceutical composition has a lower interpatient variation than an oral formulation containing d-amphetamine, and (2) having selectively prescribed the oral pharmaceutical composition; and (c) orally administering the purchased oral pharmaceutical composition to the patient.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1. Synthesis of peptide amphetamine conjugates.

FIG. 2. Synthesis of lysine amphetamine dimesylate.

FIG. 3. Synthesis of lysine amphetamine HCl.

FIG. 4. Synthesis of serine amphetamine conjugate.

FIG. 5. Synthesis of phenylalanine amphetamine conjugate.

FIG. 6. Synthesis of triglycine amphetamine conjugate.

FIG. 7. Plasma concentrations of d-amphetamine from individual rats orally administered d-amphetamine or L-lysine-d-amphetamine hydrochloride.

The following Figures (FIG. 8-FIG. 16) depict results obtained from studies of oral administration of d-amphetamine sulfate or L-lysine-d-amphetamine dimesylate to rats (ELISA analysis):

FIG. 8. Plasma concentrations of d-amphetamine (at dose 1.5 mg/kg d-amphetamine base).

FIG. 9. Plasma concentrations of d-amphetamine (at dose 3 mg/kg d-amphetamine base).

FIG. 10. Plasma concentrations of d-amphetamine (at dose 6 mg/kg d-amphetamine base).

FIG. 11. Plasma concentrations of d-amphetamine (at dose 12 mg/kg d-amphetamine base).

FIG. 12. Plasma concentrations of d-amphetamine (at dose 30 mg/kg d-amphetamine base).

FIG. 13. Plasma concentrations of d-amphetamine (at dose 60 mg/kg d-amphetamine base).

FIG. 14. Percent bioavailability (AUC and C_(max)) of L-lysine-d-amphetamine dimesylate compared to d-amphetamine sulfate at doses 1.5, 3, 6, 12, 30, and 60 mg/kg d-amphetamine base.

FIG. 15. Plasma concentrations of d-amphetamine at 30-minutes post-dose for escalating doses of d-amphetamine base.

FIG. 16. Plasma concentrations of d-amphetamine (at dose 60 mg/kg d-amphetamine base).

FIG. 17. Plasma concentrations of d-amphetamine following intranasal administration of L-lysine-d-amphetamine hydrochloride or d-amphetamine sulfate (at dose 3 mg/kg d-amphetamine base) to rats (ELISA analysis).

FIG. 18. Plasma concentrations of d-amphetamine following intranasal administration of L-lysine-d-amphetamine dimesylate or d-amphetamine sulfate (at dose 3 mg/kg d-amphetamine base) to rats (ELISA analysis).

FIG. 19. Plasma concentrations of d-amphetamine following bolus intravenous administration of L-lysine-d-amphetamine dimesylate or d-amphetamine sulfate (at dose 1.5 mg/kg d-amphetamine base) to rats (ELISA analysis).

FIG. 20. Plasma concentrations of d-amphetamine levels following oral administration of Dexedrine Spansule® capsules, crushed Dexedrine Spansule® capsules, or L-lysine-d-amphetamine dimesylate (at dose 3 mg/kg d-amphetamine base) to rats (ELISA analysis).

The following Figures (FIG. 21-FIG. 30) depict results obtained from studies of oral administration of d-amphetamine sulfate or L-lysine-d-amphetamine dimesylate to rats (LC/MS/MS analysis):

FIG. 21A and FIG. 21B. Plasma concentrations of d-amphetamine in ng/mL (FIG. 21A) and in nM (FIG. 21B) (at dose 1.5 mg/kg d-amphetamine base).

FIG. 22A and FIG. 22B. Plasma concentrations of d-amphetamine in ng/mL (FIG. 22A) and in nM (FIG. 22B) (at dose 3 mg/kg d-amphetamine base).

FIG. 23A and FIG. 23B. Plasma concentrations of d-amphetamine in ng/mL (FIG. 23A) and in nM (FIG. 23B) (at dose 6 mg/kg d-amphetamine base).

FIG. 24A and FIG. 24B. Plasma concentrations of d-amphetamine in ng/mL (FIG. 24A) and in nM (FIG. 24B) (at dose 12 mg/kg d-amphetamine base).

FIG. 25A and FIG. 25B. Plasma concentrations of d-amphetamine in ng/mL (FIG. 25A) and in nM (FIG. 25B) (at dose 60 mg/kg d-amphetamine base).

FIG. 26. Comparative bioavailability (C_(max)) of L-lysine-d-amphetamine and d-amphetamine in proportion to escalating human equivalent doses.

FIG. 27. Comparative bioavailability (AUC_(inf)) of L-lysine-d-amphetamine and d-amphetamine in proportion to escalating doses of d-amphetamine base.

FIG. 28. Comparative bioavailability (AUC_(inf)) of L-lysine-d-amphetamine and d-amphetamine in proportion to escalating human equivalent doses.

FIG. 29. Comparative bioavailability (C_(max)) of intact L-lysine-d-amphetamine in proportion to escalating human equivalent doses.

FIG. 30. Comparative bioavailability (AUC_(inf)) of intact L-lysine-d-amphetamine in proportion to escalating human equivalent doses.

FIG. 31. Plasma concentrations of d-amphetamine following intranasal administration of L-lysine-d-amphetamine dimesylate or d-amphetamine sulfate (at dose 3 mg/kg d-amphetamine base) to rats (LC/MS/MS analysis).

FIG. 32A and FIG. 32B. Plasma concentrations of d-amphetamine and L-lysine-d-amphetamine in ng/mL (FIG. 32A) and in nM (FIG. 32B), following intranasal administration of L-lysine-d-amphetamine dimesylate or d-amphetamine sulfate (at dose 3 mg/kg d-amphetamine base) to rats (LC/MS/MS analysis).

FIG. 33. Plasma concentrations of d-amphetamine following bolus intravenous administration of L-lysine-d-amphetamine dimesylate or d-amphetamine sulfate (at dose 1.5 mg/kg d-amphetamine base) to rats (LC/MS/MS analysis).

FIG. 34A and FIG. 34B. Plasma concentrations of d-amphetamine in ng/mL (FIG. 34A) and in nM (FIG. 34B), following intravenous administration of L-lysine-d-amphetamine dimesylate or d-amphetamine sulfate (at dose 1.5 mg/kg d-amphetamine base) to rats (LC/MS/MS analysis).

The following Figures (FIG. 35-FIG. 40) depict results obtained from studies of oral and intravenous administration (at dose 1 mg/kg d-amphetamine base) of d-amphetamine sulfate or L-lysine-d-amphetamine dimesylate to conscious male beagle dogs (LC/MS/MS analysis):

FIG. 35. Mean plasma concentration time profile of L-lysine-d-amphetamine following intravenous or oral administration of L-lysine-d-amphetamine (n=3).

FIG. 36. Plasma concentration time profile of d-amphetamine following intravenous or oral administration of L-lysine-d-amphetamine (n=3).

FIG. 37A and FIG. 37B. Mean plasma concentration time profile of L-lysine-d-amphetamine and d-amphetamine levels in ng/ml (FIG. 37A) and in nM (FIG. 37B), following intravenous administration of L-lysine-d-amphetamine (n=3).

FIG. 38A and FIG. 38B. Mean plasma concentration time profile of L-lysine-d-amphetamine and d-amphetamine levels in ng/ml (FIG. 38A) and in nM (FIG. 38B), following oral administration of L-lysine-d-amphetamine (n=3).

FIG. 39A and FIG. 39B. Individual plasma concentration time profile of L-lysine-d-amphetamine following intravenous administration (FIG. 39A) or oral administration (FIG. 39B) of L-lysine-d-amphetamine.

FIG. 40A and FIG. 40B. Individual plasma concentration time profile of d-amphetamine following intravenous administration (FIG. 40A) or oral administration (FIG. 40B) of L-lysine-d-amphetamine.

FIG. 41. Plasma concentrations of d-amphetamine following oral administration of L-lysine-d-amphetamine dimesylate or d-amphetamine sulfate (at dose 1.8 mg/kg d-amphetamine base) to male dogs.

FIG. 42. Plasma concentrations of d-amphetamine following oral administration of L-lysine-d-amphetamine dimesylate or d-amphetamine sulfate (at dose 1.8 mg/kg d-amphetamine base) to female dogs.

FIG. 43. Mean blood pressure following intravenous injection of increasing amounts of L-lysine-d-amphetamine dimesylate or d-amphetamine in male and female dogs.

FIG. 44. Left ventricular blood pressure following intravenous injection of increasing amounts of L-lysine-d-amphetamine dimesylate or d-amphetamine in male and female dogs.

The following Figures (FIG. 45-FIG. 49) depict results obtained from studies of oral (at dose 6 mg/kg d-amphetamine base), intranasal (at dose 1 mg/kg d-amphetamine base), and intravenous administration (at dose 1 mg/kg d-amphetamine base) of d-amphetamine sulfate or L-lysine-d-amphetamine hydrochloride to rats:

FIG. 45. Locomotor activity of rats following oral administration (5 hour time-course).

FIG. 46. Locomotor activity of rats following oral administration (12 hour time-course).

FIG. 47. Locomotor activity of rats following intranasal administration (1 hour time-course).

FIG. 48. Locomotor activity of rats following intranasal administration (with carboxymethylcellulose) (2 hour time-course).

FIG. 49. Locomotor activity of rats following intravenous administration (3 hour time-course).

The following Figures (FIG. 50-FIG. 58) depict results obtained from studies of oral, intranasal, and intravenous administration of d-amphetamine or amphetamine conjugate hydrochloride salts to rats (ELISA analysis):

FIG. 50. Intranasal bioavailability of abuse-resistant amphetamine amino acid, di-, and tri-peptide conjugates.

FIG. 51. Oral bioavailability of abuse-resistant amphetamine amino acid, di-, and tri-peptide conjugates.

FIG. 52. Intravenous bioavailability of an abuse-resistant amphetamine tri-peptide conjugate.

FIG. 53. Intranasal bioavailability of an abuse-resistant amphetamine amino acid conjugate.

FIG. 54. Oral bioavailability of an abuse-resistant amphetamine amino acid conjugate.

FIG. 55. Intravenous bioavailability of an abuse-resistant amphetamine amino acid conjugate.

FIG. 56. Intranasal bioavailability of an abuse-resistant amphetamine amino tri-peptide conjugate.

FIG. 57. Intranasal bioavailability of abuse-resistant amphetamine amino acid-, and di-peptide conjugates.

FIG. 58. Intranasal bioavailability of an abuse-resistant amphetamine di-peptide conjugate containing D- and L-amino acid isomers.

FIG. 59A and FIG. 59B. Plasma concentrations of d-amphetamine and L-lysine-d-amphetamine in ng/mL for the serum levels (FIG. 59A) and in ng/g for brain tissue (FIG. 59B), following oral administration of L-lysine-d-amphetamine hydrochloride or d-amphetamine sulfate (at dose 5 mg/kg d-amphetamine base) to rats (LC/MS/MS analysis).

FIG. 60. Steady-state plasma d-amphetamine and L-lysine-d-amphetamine levels obtained from clinical studies of oral administration of L-lysine-d-amphetamine dimesylate 70 mg to humans (LC/MS/MS analysis).

The following Figures (FIG. 61-FIG. 70) depict results obtained from clinical studies of oral administration of L-lysine-d-amphetamine dimesylate to humans (LC/MS/MS analysis):

FIG. 61A and FIG. 61B. Plasma d-amphetamine and L-lysine-d-amphetamine levels (FIG. 61A, ng/mL; FIG. 61B, nM) over a 72 hour period following oral administration of L-lysine-d-amphetamine (25 mg L-lysine-d-amphetamine dimesylate containing 7.37 mg d-amphetamine base) to humans.

FIG. 62A and FIG. 62B. Plasma d-amphetamine and L-lysine-d-amphetamine levels (FIG. 62A, ng/mL; FIG. 62B, nM) over a 72 hour period following oral administration of L-lysine-d-amphetamine (75 mg L-lysine-d-amphetamine dimesylate containing 22.1 mg d-amphetamine base) to humans.

FIG. 63A and FIG. 63B. Plasma d-amphetamine levels (FIG. 63A, 0-12 hours; FIG. 63B, 0-72 hours) following oral administration of L-lysine-d-amphetamine (75 mg L-lysine-d-amphetamine dimesylate containing 22.1 mg d-amphetamine base) or Adderall XR® (35 mg containing 21.9 mg amphetamine base) to humans.

FIG. 64A and FIG. 64B. Plasma d-amphetamine levels (FIG. 64A, 0-12 hours; FIG. 64B, 0-72 hours) following oral administration of L-lysine-d-amphetamine (75 mg L-lysine-d-amphetamine dimesylate containing 22.1 mg d-amphetamine base) or Dexedrine Spansule® (30 mg containing 22.1 mg amphetamine base) to humans.

FIG. 65. Mean plasma concentration of d-amphetamine after oral administration of single 30 mg, 50 mg, and 70 mg doses of L-lysine-d-amphetamine dimesylate under fasted conditions to pediatric patients with ADHD.

FIG. 66. Relationship between the dose-normalized AUC of d-amphetamine and gender after oral administration of L-lysine-d-amphetamine dimesylate capsules once daily to healthy adult volunteers and children with ADHD.

FIG. 67. Relationship between the dose-normalized maximum plasma concentration of d-amphetamine and gender after oral administration of L-lysine-d-amphetamine dimesylate capsules once daily to healthy adult volunteers and children with ADHD.

FIG. 68. Relationship between the dose-normalized time to maximum concentration of d-amphetamine and gender after oral administration of L-lysine-d-amphetamine dimesylate capsules once daily to healthy adult volunteers and children with ADHD.

FIG. 69. ADHD-RS at endpoint for pediatric clinical study.

FIG. 70. SKAMP score (efficacy) vs. time for pediatric clinical study.

FIGS. 71 and 72. Optical micrographs and scanning electron micrographs, respectively, of L-lysine-d-amphetamine dimesylate prepared according to the procedure described in Example 38.

FIGS. 73 and 74. Optical micrographs of L-lysine-d-amphetamine dimesylate prepared according to the procedure described in Example 39.

FIGS. 75 and 76. Scanning electron micrographs of L-lysine-d-amphetamine dimesylate prepared according to the procedure described in Example 39.

FIG. 77. X-ray powder diffractogram (XRPD) of L-lysine-d-amphetamine dimesylate prepared according to the procedure described in Example 39.

FIG. 78. Differential scanning calorimetry (DSC) thermogram of lisdexamphetamine dimesylate prepared according to the procedure described in Example 38

FIGS. 79 and 80. Fourier transform (FT)-Raman Spectra, at high and low sensitivity, of lisdexamphetamine dimesylate prepared according to the procedure described in Example 39.

FIG. 81. ORTEP drawing of the contents of the asymmetric unit of the lisdexamphetamine dimesylate crystal structure. Atoms are represented by 50% probability anisotropic thermal ellipsoids.

FIG. 82. ORTEP drawing of the two lisdexamphetamine molecules observed in the crystal structure. Atoms are represented by 50% probability anisotropic thermal ellipsoids.

FIG. 83. Packing diagram of lisdexamphetamine dimesylate viewed down the crystallographic a axis.

FIG. 84. Packing diagram of lisdexamphetamine dimesylate viewed down the crystallographic b axis.

FIG. 85. Packing diagram of lisdexamphetamine dimesylate viewed down the crystallographic c axis.

FIG. 86. Comparison of calculated XRPD spectrum from single crystal data (top) to experimental XRPD spectrum of lisdexamphetamine dimesylate prepared according to example 39.

FIG. 87. Synthesis of lysine amphetamine dimesylate.

FIG. 88 is a graph showing the mean change in SKAMP deportment score from the pre-dose measurement over 13 hours after dosing, for both placebo and L-lysine-d-amphetamine dimesylate.

FIG. 89 is a graph showing the mean change in SKAMP attention score from the pre-dose measurement over 13 hours after dosing, for both placebo and L-lysine-d-amphetamine dimesylate.

FIG. 90 is a graph showing the mean change in PERMP-A scores from the pre-dose measurement over 13 hours after dosing, for both placebo and L-lysine-d-amphetamine dimesylate.

FIG. 91 is a graph showing the mean change in PERMP-C scores from the pre-dose measurement over 13 hours after dosing, for both placebo and L-lysine-d-amphetamine dimesylate.

FIG. 92 is a histogram analysis of CGI-I scores for a cross-over treatment phase (between lysine-d-amphetamine dimesylate and placebo).

FIG. 93 is a graph showing the mean plasma d-amphetamine concentration over time, for individual subjects.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides amphetamine prodrugs comprising amphetamine covalently bound to a chemical moiety. The amphetamine prodrugs can also be characterized as conjugates in that they possess a covalent attachment. They may also be characterized as conditionally bioreversible derivatives (“CBDs”) in that the amphetamine prodrug preferably remains inactive until oral administration releases the amphetamine from the chemical moiety.

In one embodiment, the invention provides an amphetamine prodrug of Formula I:

A-X_(n)-Z_(m)  (I)

wherein A is an amphetamine;

each X is independently a chemical moiety;

each Z is independently a chemical moiety that acts as an adjuvant and is different from at least one X;

n is an increment from 1 to 50, preferably 1 to 10; and

m is an increment from 0 to 50, preferably 0.

When m is 0, the amphetamine prodrug is a compound of Formula (II):

A-X_(n)  (II)

wherein each X is independently a chemical moiety.

Formula (II) can also be written to designate the chemical moiety that is physically attached to the amphetamine:

A-X₁-(X)_(n-1)  (III)

wherein A is an amphetamine; X₁ is a chemical moiety, preferably a single amino acid; each X is independently a chemical moiety that is the same as or different from X₁; and n is an increment from 1 to 50.

The amphetamine, A, can be any of the sympathomimetic phenethylamine derivatives which have central nervous system stimulant activity such as amphetamine, or any derivative, analog, or salt thereof. Exemplary amphetamines include, but are not limited to, amphetamine, methamphetamine, methylphenidate, p-methoxyamphetamine, methylenedioxyamphetamine, 2,5-dimethoxy-4-methylamphetamine, 2,4,5-trimethoxyamphetamine, and 3,4-methylenedioxymethamphetamine, N-ethylamphetamine, fenethylline, benzphetamine, and chlorphentermine as well as the amphetamine compounds of Adderall®; actedron; actemin; adipan; akedron; allodene; alpha-methyl-(±)-benzeneethanamine; alpha-methylbenzeneethanamine; alpha-methylphenethylamine; amphetamine; amphate; anorexine; benzebar; benzedrine; benzyl methyl carbinamine; benzolone; beta-amino propylbenzene; beta-phenylisopropylamine; biphetamine; desoxynorephedrine; dietamine; DL-amphetamine; elastonon; fenopromin; finam; isoamyne; isomyn; mecodrin; monophos; mydrial; norephedrane; novydrine; obesin; obesine; obetrol; octedrine; oktedrin; phenamine; phenedrine; phenethylamine, alpha-methyl-; percomon; profamina; profetamine; propisamine; racephen; raphetamine; rhinalator, sympamine; simpatedrin; simpatina; sympatedrine; and weckamine. Preferred amphetamines include methamphetamine, methylphenidate, and amphetamine, with amphetamine being most preferred.

The amphetamine can have any stereogenic configuration, including both dextro- and levo-isomers. The dextro-isomer, particularly dextroamphetamine, is preferred.

Preferably, the amphetamine is an amphetamine salt. Pharmaceutically acceptable salts, e.g., non-toxic, inorganic and organic acid addition salts, are known in the art. Exemplary salts include, but are not limited to, 2-hydroxyethanesulfonate, 2-naphthalenesulfonate, 3-hydroxy-2-naphthoate, 3-phenylpropionate, acetate, adipate, alginate, amsonate, aspartate, benzenesulfonate, benzoate, besylate, bicarbonate, bisulfate, bitartrate, borate, butyrate, calcium edetate, camphorate, camphorsulfonate, camsylate, carbonate, citrate, clavulariate, cyclopentanepropionate, digluconate, dodecylsulfate, edetate, edisylate, estolate, esylate, ethanesulfonate, finnarate, gluceptate, glucoheptanoate, gluconate, glutamate, glycerophosphate, glycollylarsanilate, hemisulfate, heptanoate, hexafluorophosphate, hexanoate, hexylresorcinate, hydrabamine, hydrobromide, hydrochloride, hydroiodide, hydroxynaphthoate, iodide, isothionate, lactate, lactobionate, laurate, laurylsulphonate, malate, maleate, mandelate, mesylate, methanesulfonate, methylbromide, methylnitrate, methylsulfate, mucate, naphthylate, napsylate, nicotinate, nitrate, N-methylglucamine ammonium salt, oleate, oxalate, palmitate, pamoate, pantothenate, pectinate, persulfate, phosphate, phosphateldiphosphate, picrate, pivalate, polygalacturonate, propionate, p-toluenesulfonate, saccharate, salicylate, stearate, subacetate, succinate, sulfate, sulfosaliculate, suramate, tannate, tartrate, teoclate, thiocyanate, tosylate, triethiodide, undecanoate, and valerate salts, and the like. (See Berge et al. (1977) “Pharmaceutical Salts”, J. Pharm. Sci. 66:1-19). A preferred amphetamine salt is the mesylate salt (e.g., as in L-lysine-d-amphetamine dimesylate).

Particular salts may be less hygroscopic thereby facilitating handling. In a preferred embodiment, the amphetamine prodrug has a water content (Karl Fischer analysis) of about 0% to about 5%, about 0.1% to about 3%, about 0.25% to about 2%, or increments therein. When the amphetamine prodrug is formulated into a pharmaceutical composition, the pharmaceutical composition preferably has a water content of about 1% to about 10%, about 1% to about 8%, about 2% to about 7%, or increments therein.

DEFINITIONS

The term “crystal” refers to a form of a solid state of matter, which is distinct from its amorphous solid state. Crystals display characteristic features including a lattice structure, characteristic shapes and optical properties such as refractive index. A crystal consists of atoms arranged in a pattern that repeats periodically in three dimensions.

An “anhydrous crystal form” lacks bound water molecules.

The term “polymorph” refers to crystallographically distinct forms of a substance.

The term “amine” refers to a —NH₂ group.

The D₁₀, D₅₀ and D₉₀ represent the 10^(th) percentile, median or the 50th percentile and the 90th percentile of the particle size distribution, respectively, as measured by diameter. That is, the D₁₀ (D₅₀, D₉₀) is a value on the distribution such that 10% (50%, 90%) of the particles have a volume of this value or less.

An “effective amount of drug” is an amount of lisdexamphetamine dimesylate which is effective to treat or prevent a condition in a living organism to whom it is administered over some period of time, e.g., provides a therapeutic effect during a desired dosing interval.

The term “treating” includes: (1) preventing or delaying the appearance of clinical symptoms of the state, disorder or condition developing in an animal that may be afflicted with or predisposed to the state, disorder or condition but does not yet experience or display clinical or subclinical symptoms of the state, disorder or condition; (2) inhibiting the state, disorder or condition (i.e., arresting, reducing or delaying the development of the disease, or a relapse thereof in case of maintenance treatment, of at least one clinical or subclinical symptom thereof); and/or (3) relieving the condition (i.e., causing regression of the state, disorder or condition or at least one of its clinical or subclinical symptoms). The benefit to a patient to be treated is either statistically significant or at least perceptible to the patient or to the physician.

As used herein in connection with a measured quantity, the term “about” refers to the normal variation in that measured quantity that would be expected by the skilled artisan making the measurement and exercising a level of care commensurate with the objective of the measurement and the precision of the measuring equipment used. Unless otherwise indicated, “about” refers to a variation of ±10% of the value provided.

Throughout this application, the term “increment” is used to define a numerical value in varying degrees of precision, e.g., to the nearest 10, 1, 0.1, 0.01, etc. The increment can be rounded to any measurable degree of precision. For example, the range 1 to 100 or increments therein includes ranges such as 20 to 80, 5 to 50, 0.4 to 98, and 0.04 to 98.05.

The amphetamine is bound to one or more chemical moieties, denominated X and Z. A chemical moiety can be any moiety that decreases the pharmacological activity of amphetamine while bound to the chemical moiety as compared to unbound (free) amphetamine. The attached chemical moiety can be either naturally occurring or synthetic. Exemplary chemical moieties include, but are not limited to, peptides, including single amino acids, dipeptides, tripeptides, oligopeptides, and polypeptides; glycopeptides; carbohydrates; lipids; nucleosides; nucleic acids; and vitamins. Preferably, the chemical moiety is generally recognized as safe (“GRAS”).

“An extended release mixed amphetamine composition,” such as Adderall™ XR, is a composition that contains at least 2 amphetamine salts. The composition allows for extended release of amphetamine into the bloodstream.

“Carbohydrates” include sugars, starches, cellulose, and related compounds, e.g., (CH₂O)_(n) wherein n is an integer larger than 2, and C_(n)(H₂O)_(n-1) wherein n is an integer larger than 5. The carbohydrate can be a monosaccharide, disaccharide, oligosaccharide, polysaccharide, or a derivative thereof (e.g., sulfo- or phospho-substituted). Exemplary carbohydrates include, but are not limited to, fructose, glucose, lactose, maltose, sucrose, glyceraldehyde, dihydroxyacetone, erythrose, ribose, ribulose, xylulose, galactose, mannose, sedoheptulose, neuraminic acid, dextrin, and glycogen.

A “glycopeptide” is a carbohydrate linked to an oligopeptide. Similarly, the chemical moiety can also be a glycoprotein, glyco-amino-acid, or glycosyl-amino-acid. A “glycoprotein” is a carbohydrate (e.g., a glycan) covalently linked to a protein. A “glyco-amino-acid” is a carbohydrate (e.g., a saccharide) covalently linked to a single amino acid. A “glycosyl-amino-acid” is a carbohydrate (e.g., a saccharide) linked through a glycosyl linkage (O—, N—, or S—) to an amino acid.

A “peptide” includes a single amino acid, a dipeptide, a tripeptide, an oligopeptide, a polypeptide, or a carrier peptide. An oligopeptide includes from 2 to 70 amino acids.

The term “amino acid” includes both naturally occurring (i.e., the 20 amino acids used for protein synthesis) and nonnaturally occurring amino acids, synthetically produced amino acids.

Preferably, the chemical moiety is a peptide, more particularly a single amino acid, a dipeptide, or a tripeptide. The peptide preferably comprises fewer than 70 amino acids, fewer than 50 amino acids, fewer than 10 amino acids, or fewer than 4 amino acids. When the chemical moiety is one or more amino acids, the amphetamine is preferably bound to lysine, serine, phenylalanine, or glycine. In another embodiment, the amphetamine is preferably bound to lysine, glutamic acid, or leucine. In one embodiment, the amphetamine is bound to lysine and optional additional chemical moieties, e.g., additional amino acids. In a preferred embodiment, the amphetamine is bound to a single lysine amino acid.

In one embodiment, the chemical moiety is from 1 to 12 amino acids, preferably 1 to 8 amino acids. In another embodiment, the number of amino acids is 1, 2, 3, 4, 5, 6, or 7. In another embodiment, the molecular weight of the chemical moiety is below about 2,500 kD, more preferably below about 1,000 kD, and most preferably below about 500 kD.

Each amino acid can be any one of the L- or D-enantiomers, preferably L-enantiomers, of the naturally occurring amino acids: alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartic acid (Asp or D), cysteine (Cys or C), glycine (Gly or G), glutamic acid (Glu or E), glutamine (Gln or Q), histidine (His or H), isoleucine (Ile or I), leucine (Leu or L), lysine (Lys or K), methionine (Met or M), proline (Pro or P), phenylalanine (Phe or F), serine (Ser or S), tryptophan (Trp or W), threonine (Thr or T), tyrosine (Tyr or Y), and valine (Val or V). In a preferred embodiment, the peptide comprises only naturally occurring amino acids and/or only L-amino acids. Each amino acid can be an unnatural, non-standard, or synthetic amino acids, such as aminohexanoic acid, biphenylalanine, cyclohexylalanine, cyclohexylglycine, diethylglycine, dipropylglycine, 2,3-diaminoproprionic acid, homophenylalanine, homoserine, homotyrosine, naphthylalanine, norleucine, ornithine, phenylalanine (4-fluoro), phenylalanine(2,3,4,5,6-pentafluoro), phenylalanine(4-nitro), phenylglycine, pipecolic acid, sarcosine, tetrahydroisoquinoline-3-carboxylic acid, and tert-leucine. Preferably, synthetic amino acids with alkyl side chains are selected from C₁-C₁₇ alkyls, preferably C₁-C₆ alkyls. In one embodiment, the peptide comprises one or more amino acid alcohols, e.g., serine and threonine. In another embodiment, the peptide comprises one or more N-methyl amino acids, e.g., N-methyl aspartic acid.

In one embodiment, the peptides are utilized as base short chain amino acid sequences and additional amino acids are added to the terminus or side chain. In another embodiment, the peptide may have an one or more amino acid substitutions. Preferably, the substitute amino acid is similar in structure, charge, or polarity to the replaced amino acid. For instance, isoleucine is similar to leucine, tyrosine is similar to phenylalanine, serine is similar to threonine, cysteine is similar to methionine, alanine is similar to valine, lysine is similar to arginine, asparagine is similar to glutamine, aspartic acid is similar to glutamic acid, histidine is similar to proline, and glycine is similar to tryptophan.

The peptide can comprise a homopolymer or heteropolymer of naturally occurring or synthetic amino acids. For example, the side chain attachment of amphetamine to the peptide can be a homopolymer or heteropolymer containing glutamic acid, aspartic acid, serine, lysine, cysteine, threonine, asparagine, arginine, tyrosine, or glutamine.

Exemplary peptides include Lys, Ser, Phe, Gly-Gly-Gly, Leu-Ser, Leu-Glu, homopolymers of Glu and Leu, and heteropolymers of (Glu)_(n)-Leu-Ser. In a preferred embodiment, the peptide is Lys, Ser, Phe, or Gly-Gly-Gly.

In one embodiment, the chemical moiety has one or more free carboxy and/or amine terminal and/or side chain group other than the point of attachment to the amphetamine. The chemical moiety can be in such a free state, or an ester or salt thereof.

The chemical moiety can be covalently attached to the amphetamine either directly or indirectly through a linker. Covalent attachment may comprise an ester or carbonate bond. The site of attachment typically is determined by the functional group(s) available on the amphetamine. For example, a peptide can be attached to an amphetamine via the N-terminus, C-terminus, or side chain of an amino acid. For additional methods of attaching amphetamine to various exemplary chemical moieties, see U.S. application Ser. No. 10/156,527, PCT/US03/05524, and PCT/US03/05525, each of which is hereby incorporated by reference in its entirety.

Synthesis of Lisdexamphetamine and Salts Thereof.

Lisdexamphetamine and salts thereof can be prepared from L-lysine or a salt thereof as follows. The amine groups on the L-lysine or a salt thereof are protected, for example, by reaction with di-tert-butyl dicarbonate. Suitable amine protecting groups include, but are not limited to, Boc (—C(O)OC(CH₃)₃), Boc (tert-butyloxycarbonyl, —C(O)OC(CH₃)₃), Cbz (benzyloxycarbonyl, —C(O)OCH₂Ph), Alloc (allyloxycarbonyl, —C(O)OCH₂CH═CH₂), Fmoc (Fluorenylmethyloxycarbonyl, —C(O)OCH₂-fluorene) or Teoc (Trimethylsilylethyl-oxycarbonyl, —C(O)OCH₂CH₂Si(CH₃)₃). In addition, amine protecting groups given in J. Chem. Soc., Perkin Trans. Vol. 1, 1998, p. 4005-4037, hereby incorporated by reference in its entirety. Preferably this reaction is performed in the presence of a base, such as sodium hydroxide.

The di-amine protected L-lysine is then subjected to an acid activation followed by an amidation reaction with d-amphetamine to form a di-amine protected lisdexamphetamine. The amidation reaction can be performed either by first activating the acid group of the protected L-lysine, and then amidating the activated acid by, for example, reacting it with d-amphetamine (“two-step procedure”), or in a “one-pot procedure” in which the protected L-lysine, d-amphetamine, coupling reagent, and optional additive are all combined simultaneously. For the two-step procedure, the acid group of the protected L-lysine can be activated, for example, by reaction with DCC (dicyclohexylcarbodiimide), CDI (carbonyldiimidazole), EDCI (1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide), PyBOP (benzotriazol-1-yloxytris(pyrrolidino)phosphonium hexafluorophosphate), or HBTU (O-(1H-benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate) in addition to, for example, N-hydroxysuccinimide, N-hydroxybenzotriazole, or a phenol such as p-nitrophenol, or by reaction with, for instance, iso-butylchloroformate. Preferably, the activated ester is then isolated before being allowed to react with d-amphetamine.

In the one-pot procedure, the activation of the protected L-lysine acid and subsequent amidation can be performed by mixing the protected L-lysine, d-amphetamine, coupling reagent and optional additive simultaneously. The coupling agent may be, for example, DCC (dicyclohexylcarbodiimide), CDI (carbonyldiimidazole), EDCI (1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide), PyBOP (benzotriazol-1-yloxytris(pyrrolidino)phosphonium hexafluorophosphate), or HBTU (O-(1H-benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate). The optional additive may be, for example, N-hydroxysuccinimide, N-hydroxybenzotriazole, or a phenol such as p-nitrophenol. Preferably, the acid activation step is performed in the presence of a dehydration agent, such as dicyclohexylcarbodiimide (DCC). In either the two-step or one one-pot method, the amidating step can be performed in the presence of an organic base, such as N-methyl-morpholine (NMM).

The di-amine protected lisdexamphetamine is then deprotected and optionally converted to a salt. The deprotection and salt conversion can be performed in a single reaction. For example, the di-amine protected lisdexamphetamine can be reacted with methane sulfonic acid to form lisdexamphetamine dimesylate. Preferably, this reaction is performed in the presence of an alcohol, such as isopropanol.

The amphetamine prodrug compounds described above can be synthesized as described in Examples 1, 38 and 39 and FIG. 1 and FIG. 87. Preferably, additional purification and/or crystallization steps are not necessary to yield a highly pure product. In one embodiment, the purity of the amphetamine prodrug is at least about 95%, more preferably at least about 96%, 97%, 98%, 98.5%, 99%, 99.5%, 99.9%, or increments therein. For the synthesis of L-lysine-d-amphetamine, known impurities include Lys-Lys-d-amphetamine, Lys(Lys)-d-amphetamine, d-amphetamine, Lys(Boc)-d-amphetamine, Boc-Lys-d-amphetamine, and Boc-Lys(Boc)-d-amphetamine. In one embodiment, the presence of any single impurity is less than about 3%, more preferably less than about 2%, 1%, 0.5%, 0.25%, 0.2%, 0.15%, 0.1%, 0.05%, or increments therein (e.g., greater than 0% but less than 0.2, 0.1, or 0.05%). The impurities and their structures are given in the table below.

Common Name IUPAC Name Structure Lys(Lys)- Dex or LysLysDex Isomer 1 (2S)-2,6-Diamino-N-{(5S)- 5-amino-6-[[(1S)-1- methyl-2- phenylethyl]amino]-6- oxohexyl}hexanamide trimethanesulfonate

LysLys-Dex or LysLysDex Isomer 2 (2S)-2,6-Diamino-N-{(1S)- 5-amino-1-[[((1S)-1- methyl-2- phenylethyl)amino]carbon yl]pentyl} hexanamide trimethanesulfonate

BocLys-Dex or (monoBoc)L ys-Dex 1 tert-Butyl (1S)-5-amino-1- {[((1S)-1-methyl-2- phenylethyl)amino]carbon yl}pentylcarbamate methanesulfonate

Lys(Boc)- Dex or (monoBoc)L ys-Dex 2 tert-Butyl (5S)-5-amino-6- {[(1S)-1-methyl-2- phenylethyl]amino}-6- oxohexylcarbamate methanesulfonate

Boc- Lys(Boc)- Dex tert-Butyl (1S)-5-[(tert- butoxycarbonyl)amino]-1- ({[(1S)-1-methyl-2- phenylethyl]amino}carbonyl) pentylcarbamate

Diastereomer (2R)-2,6-Diamino-N-[(1S)- 1-methyl-2- phenylethyl]hexanamide dimethanesulfonate

Dextroamphetamine methanesulfonate (1S)-1-Methyl-2- phenylethylamine methanesulfonate

In one embodiment, the amphetamine prodrug (a compound of one of the formulas described above) may exhibit one or more of the following advantages over free amphetamines. The amphetamine prodrug may prevent overdose by exhibiting a reduced pharmacological activity when administered at higher than therapeutic doses, e.g., higher than the prescribed dose. Yet when the amphetamine prodrug is administered at therapeutic doses, the amphetamine prodrug may retain similar pharmacological activity to that achieved by administering unbound amphetamine, e.g., Adderall XR®. Also, the amphetamine prodrug may prevent abuse by exhibiting stability under conditions likely to be employed by illicit chemists attempting to release the amphetamine. The amphetamine prodrug may prevent abuse by exhibiting reduced bioavailability when it is administered via parenteral routes, particularly the intravenous (“shooting”), intranasal (“snorting”), and/or inhalation (“smoking”) routes that are often employed in illicit use. Thus, the amphetamine prodrug may reduce the euphoric effect associated with amphetamine abuse. Thus, the amphetamine prodrug may prevent and/or reduce the potential of abuse and/or overdose when the amphetamine prodrug is used in a manner inconsistent with the manufacturer's instructions, e.g., consuming the amphetamine prodrug at a higher than therapeutic dose or via a non-oral route of administration.

Use of phrases such as “decreased”, “reduced”, “diminished”, or “lowered” includes at least a 10% change in pharmacological activity with greater percentage changes being preferred for reduction in abuse potential and overdose potential. For instance, the change may also be greater than 25%, 35%, 45%, 55%, 65%, 75%, 85%, 95%, 96%, 97%, 98%, 99%, or other increments greater than 10%.

The term “bioavailability” refers to the rate and extent to which a drug is absorbed. One measurement of bioavailability is defined by the fraction (F) of the dose that reaches systemic circulation. Thus, in extreme cases, F=0 in drugs which are not absorbed at all in the GI tract while for drugs that are completely absorbed (and not metabolized by a first pass effect) F=1. The bioavailability can also be calculated from the area under the curve (AUC) of the serum level vs. time plot.

The coefficient of variation (CV) is typically used to express the variability in bioavailability. This value is obtained by expressing the standard deviation as a percentage of the arithmetic mean.

Use of the phrase “similar pharmacological activity” means that two compounds exhibit curves that have substantially the same AUC, C_(max), T_(max), C_(min), and/or t_(1/2) parameters, preferably within about 30% of each other, more preferably within about 25%, 20%, 10%, 5%, 2%, 1%, or other increments less than 30%.

Preferably, the amphetamine prodrug exhibits an unbound amphetamine oral bioavailability of at least about 60% AUC (area under the curve), more preferably at least about 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or other increments greater than 60%. Preferably, the amphetamine prodrug exhibits an unbound amphetamine parenteral, e.g., intranasal, bioavailability of less than about 70% AUC, more preferably less than about 50%, 30%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, or other increments less than 70%. For certain treatments, it is desirable that the amphetamine prodrug exhibits both the oral and parenteral bioavailability characteristics described above. See, e.g., Table 61.

Preferably, the amphetamine prodrug remains inactive until oral administration releases the amphetamine. Without being bound by theory, it is believed that the amphetamine prodrug is inactive because the attachment of the chemical moiety reduces binding between the amphetamine and its biological target sites (e.g., human dopamine (“DAT”) and norepinephrine (“NET”) transporter sites). (See Hoebel, B. G., L. Hernandez, et al., “Microdialysis studies of brain norepinephrine, serotonin, and dopamine release during ingestive behavior, Theoretical and clinical implications.” Ann NY Acad Sci 575: 171-91 (1989)). The chemical moiety attachment may reduce binding between amphetamine and DAT and/or NET in part because the amphetamine prodrug cannot cross the blood-brain barrier. The amphetamine prodrug is activated by oral administration, that is, the amphetamine is released from the chemical moiety by hydrolysis, e.g., by enzymes in the stomach, intestinal tract, or blood serum. Because oral administration facilitates activation, activation is reduced when the amphetamine prodrug is administered via parenteral routes often employed by illegal users.

Further, it is believed that the amphetamine prodrug is resistant to abuse and/or overdose due to a natural gating mechanism at the site of hydrolysis, namely the gastrointestinal tract. This gating mechanism is thought to allow the release of therapeutic amounts of amphetamine from the amphetamine prodrug, but limit the release of higher amounts of amphetamine.

In another embodiment, the toxicity of the amphetamine prodrug is substantially lower than that of the unbound amphetamine. For example, in a preferred embodiment, the acute toxicity is 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold less, or increments therein less lethal than oral administration of unbound amphetamine.

Preferably, the amphetamine prodrug provides a serum release curve that does not increase above amphetamine's toxicity level when administered at higher than therapeutic doses. The amphetamine prodrug may exhibit a reduced rate of amphetamine absorption and/or an increased rate of clearance compared to the free amphetamine. The amphetamine prodrug may also exhibit a steady-state serum release curve. Preferably, the amphetamine prodrug provides bioavailability but prevents C_(max) spiking or increased blood serum concentrations. Pharmacokinetic parameters are described in the Examples below, particularly the clinical pharmacokinetic Examples. In one embodiment, the amphetamine prodrug provides similar pharmacological activity to the clinically measured pharmacokinetic activity of L-lysine-d-amphetamine dimesylate. For example, the pharmacological parameters (AUC, C_(max), T_(max), C_(min), and/or t_(1/2)) are preferably within 80% to 125%, 80% to 120%, 85% to 125%, 90% to 110%, or increments therein, of the given values. It should be recognized that the ranges can, but need not be symmetrical, e.g., 85% to 105%. For the pediatric study, the pharmacokinetic parameters of d-amphetamine released from L-lysine-d-amphetamine dimesylate are listed in Table 72.

The amphetamine prodrug may exhibit delayed and/or sustained release characteristics. Delayed release prevents rapid onset of pharmacological effects, and sustained release is a desirable feature for particular dosing regimens, e.g., once a day regimens. The amphetamine prodrug may achieve the release profile independently. Alternatively, the amphetamine prodrug may be pharmaceutically formulated to enhance or achieve such a release profile. It may be desirable to reduce the amount of time until onset of pharmacological effect, e.g., by formulation with an immediate release product.

Accordingly, the invention also provides methods comprising providing, administering, prescribing, or consuming an amphetamine prodrug. The invention also provides pharmaceutical compositions comprising an amphetamine prodrug. The formulation of such a pharmaceutical composition can optionally enhance or achieve the desired release profile.

In one embodiment, the invention provides methods for treating a patient comprising administering a therapeutically effective amount of an amphetamine prodrug or salt thereof, i.e., an amount sufficient to prevent, ameliorate, and/or eliminate the symptoms of a disease. These methods can be used to treat any disease that may benefit from amphetamine-type drugs including, but not limited to: attention deficit disorders, e.g., ADD and ADHD, and other learning disabilities; obesity; Alzheimer's disease, amnesia, and other memory disorders and impairments; fibromyalgia; fatigue and chronic fatigue; depression; epilepsy; obsessive compulsive disorder (OCD); oppositional defiant disorder (ODD); anxiety; resistant depression; stroke rehabilitation; Parkinson's disease; mood disorder; schizophrenia; Huntington's disorder; dementia, e.g., AIDS dementia and frontal lobe dementia; movement disfunction; apathy; Pick's disease; Creutzfeldt-Jakob disease, sleep disorders, e.g., narcolepsy, cataplexy, sleep paralysis, cancer related fatigue, excessive daytime sleepiness in patients suffering from obstructive sleep apnea and hypnagogic hallucinations; conditions related to brain injury or neuronal degeneration, e.g., multiple sclerosis, Tourette's syndrome, and impotence; and nicotine dependence and withdrawal. Preferred indications include ADD, ADHD, narcolepsy, and obesity, with ADHD being most preferred.

In a further embodiment, the amphetamine prodrug to be administered comprises amphetamine covalently bound to a peptide comprising 1 to 10 amino acids. The prodrug can also be administered in a pharmaceutical composition. A preferred prodrug is L-lysine-d-amphetamine. A preferred prodrug salt is L-lysine-d-amphetamine dimesylate.

Cancer Related Fatigue

In one embodiment, the prodrugs (or pharmaceutical salts thereof) are administered to treat cancer related fatigue. The term “cancer related fatigue” includes a patient's fatigue during the period in which the patient is undergoing chemotherapy and/or radiation treatment as well as a patient's fatigue while not undergoing any such treatment (e.g., after a patient has undergone chemotherapy and/or radiation treatment).

An effective amount of the prodrug (or pharmaceutically acceptable salt thereof) is administered. For example, a daily dose of 5-100 mg of the prodrug can be administered. According to one embodiment, a daily dose of 5 or 10 mg of the prodrug is administered and is optionally titrated every 1 to 7, e.g., 3-5 days (for example, 3 or 5 days), by 5 or 10 mg increments, to the desired level of treatment. The maximum level of treatment is preferably 70 mg. Alternatively, the dose can be held constant at 5 or 10 mg as well. Preferably, the daily dose is administered once daily.

Excessive Daytime Sleepiness in Patients Suffering from Obstructive Sleep Apnea

In one embodiment, the prodrugs (or pharmaceutical salts thereof) are administered to treat excessive daytime sleepiness in patients suffering from obstructive sleep apnea. An effective amount of the prodrug (or pharmaceutically acceptable salt thereof) is administered. For example, a daily dose of 5-100 mg of the prodrug can be administered. According to one embodiment, a daily dose of 5 or 10 mg of the prodrug is administered and is optionally titrated every 3-5 days by 5 or 10 mg increments, to the desired level of treatment. The maximum level of treatment is preferably 70 mg. Alternatively, the dose can be held constant at 5 or 10 mg as well. Preferably, the daily dose is administered once daily.

Fatigue related to cancer, excessive daytime sleepiness in patients suffering from obstructive sleep apnea, or fatigue related to any other sleeping disorders can be measured by various instruments. A non-exhaustive list of instruments to measure fatigue include the Piper Fatigue Scale (Piper, B F, et al., Oncol. Nurs. Forum, vol 25, pp. 677-84 (1998)), Lee Fatigue Scale (Lee, K A, et al., Psychiatry Res., vol. 36, pp. 297-8 (1991)), Functional Assessment of Cancer Therapy-Anemia/Fatigue (Yellen, S B, J. Pain Symptom Manage., vol. 13, pp. 63-74 (1997)), Brief Fatigue Inventory (Mendoza, T R, Cancer, vol 85, pp. 1186-96 (1999)), Cancer Fatigue Scale (Okuyama, T, J. Pain Symptom Manage., vol. 19, pp. 5-14 (2000)), Schwartz Cancer Fatigue Scale (Schwartz, A L, Oncol. Nurs. Forum, vol 25, pp. 711-7 (1998)), Multidimensional Fatigue Inventory (Smets, E M, J. Psychosom. Res., vol 39, pp. 315-25 (1995)), Fatigue Severity Scale (Krupp, L B, et al., Arch Neurol., vol. 46, pp. 1121-3), Multidimensional Characterization of Fatigue Measure (Vercoulen, J H, et al., Arch. Of Neurol., vol. 53, pp. 642-49 (1996)), and The Pittsburgh Sleep Quality Index (PSQI) (Buysse, D J, et al., Psychiatry Res., vol. 2, pp. 193-213 (1989)).

These studies employ self-reporting measures, which are compared to controls lacking fatigue, result in a score which can be compared before and after treatment to assess the improvement in fatigue symptoms following treatment with the prodrug.

For example, the Pittsburgh Sleep Quality Index (PSQI) can be used to determine whether the prodrug has an effect on cancer related fatigue, or any other fatigue or sleep related disorder, including excessive daytime sleepiness in patients suffering from obstructive sleep apnea. The PSQI is a questionnaire which assesses sleep quality and disturbances over a 1-month time interval. Nineteen individual items generate seven component scores: subjective sleep quality, sleep latency, sleep duration, habitual sleep efficiency, sleep disturbances, use of sleeping medication, and daytime dysfunction. The sum of the scores for these seven components yields one global score (Buysse, D J, et al., Psychiatry Res., vol. 2, pp. 193-213 (1989)).

Combination Therapies

The methods of treatment include combination therapies which further comprise administering one or more therapeutic agents in addition to administering an amphetamine prodrug (or pharmaceutically acceptable salt thereof). In these embodiments, the therapeutic agents can be administered serially or together.

In one embodiment, the prodrugs of the present invention (or pharmaceutically acceptable salts thereof) are used in combination with another antidepressant for adjunctive antidepressant therapy.

The active ingredients can be formulated into a single dosage form, or they can be formulated together or separately among multiple dosage forms. The active ingredients can be administered simultaneously or sequentially in any order. Exemplary combination therapies include the administration of the drugs listed in Table 1.

The prodrug (or pharmaceutical salts thereof) can be administered in combination with an antidepressant to treat depression or a depressive disorder, such as major depressive disorder. The prodrug and antipressant can be can be administered serially (in any order) or together (simultaneously). The active ingredients can be formulated into a single dosage form, or they can be formulated together or separately among multiple dosage forms.

In the adjunctive antidepressant therapy embodiment, the prodrugs of the present invention, and specifically the preferred prodrug salt l-lysine-d-amphetamine dimesylate, can be paired with various antidepressants for co-therapy, including, but not limited to, serotonin norepinephrine reuptake inhibitors (SNRIs) (e.g., venlafaxine), selective serotonin reuptake inhibitors (SSRIs), and tertiary amine tricyclic norepinephrine reuptake inhibitors. For example, tertiary amine tricyclic norepinephrine reuptake inhibitors such as amitriptyline, clomipramine, doxepin, imipramine and (+)-trimipramine can be employed with l-lysine-d-amphetamine or a pharmaceutically acceptable salt thereof (e.g., the dimesylate salt).

In another adjunctive antidepressant therapy embodiment, secondary amine tricyclics such as amoxapine, desipramine, maprotiline, nortriptyline and protriptyline are paired with an amphetamine prodrug for adjunctive antidepressant therapy. The antidepressant can also be a selective serotonin reuptake inhibitor, such as citalopram, escitalopram, fluoxetine, fluvoxamine, paroxetine, or sertraline.

In yet another adjunctive antidepressant therapy embodiment, a prodrug of the present invention is administered with an atypical antidepressant. Non-limiting examples of such compounds include atomoxetine, bupropion, duloxetine, nefazodone and trazadone.

Monoamine oxidase inhibitors can also be used in combination with the prodrugs of the present invention for adjunctive antidepressant therapy. Examples include phenelzine, tranylcypromine and selegiline.

TABLE 1 Exemplary drug therapies contemplated for use in combination with an amphetamine prodrug Condition Exemplary drug class Specific exemplary drugs ADHD Amphetamine Ritalin ®, Dexedrine ®, Adderall ®, Cylert ®, Clonidine, Guanfacine Alzheimer's disease Reminyl ®, Cognex ®, Aricept ®, Exelon ®, Akatinol ®, Neotropin, Eldepryl ®, Estrogen, Clioquinol, Ibuprofen, Ginko Biloba Anxiety Antidepressant (SSRI, Elavil, Asendin ®, benzodiazepine, MAOI), Wellbutrin ®, Tegretol ®, anxiolytic Anafranil ®, Norpramine ®, Adapin ®, Sinequan ®, Tofranil ®, Epitol ®, Janimire ®, Pamelor ®, Ventyl ®, Aventyl ®, Surmontil ®, Prozac ®, Luvox ®, Serzone ®, Paxil ®, Zoloft ®, Effexor ®, Xanax ®, Librium ®, Klonopin ®, Valium ®, Zetran ®, Valrelease ®, Dalmane ®, Ativan ®, Alzapam ®, Serax ®, Halcion ®, Aurorix ®, Manerix ®, Nardil ®, Parnate ®. Apathy Amisulpride, Olanzapine, Visperidone, Quetiapine, Clozapine, Zotepine Cataplexy Xyrem ® Dementia Thioridazine, Haloperidol, Risperidone, Cognex ®, Aricept ®, Exelon ® Depression Antidepressant Fluoxetine (e.g., Prozac ®), Zoloft ®, Paxil ®, Reboxetine, Wellbutrin ®, Olanzapine, Elavil ®, Totranil ®, Pamelor ®, Nardil ®, Parnate ®, Desyrel ®, Effexor ® Fatigue Benzodiazepine Anaprox ®, Naprosen, Prozac ®, Zoloft ®, Paxil ®, Effexor ®, Desyrel ® Fibromyalgia Non-steroidal anti- Dilantin ®, Carbatrol ®, inflammatory drugs Epitol ®, Tegretol ®, Depacon ®, Depakote ®, Norpramin ®, Aventyl ®, Pamelor ®, Elavil ®, Enovil ®, Adapin ®, Sinequan ®, Zonalon ® Hallucinations Clozapine, Risperidone, Zyprexa ®, Seroquel ® Huntington's disorder Haloperidol, Clonzepam Narcolepsy Modafinil (e.g., Provigil ®), Dexedrine ®, Ritalin ® Mood disorder Thorazine ®, Haldol ®, Navane ®, Mellaril ®, Clozaril ®, Risperidone (e.g., Risperdal ®), Olanzapine (e.g., Zyprexa ®), Clozapine Obsessive-compulsive SSRI Anafranil ®, Prozac ®, disorder (OCD) Zoloft ®, Paxil ®, Luvox ® Oppositional defiant Clonidine, Risperidone, disorder (ODD) Zyprexa ®, Wellbutrin ®, Parkinson's disease Levodopa, Parlodel ®, Permax ®, Mirapex ® Schizophrenia Clozapine, Zyprexa ®, Seroquel ®, and Risperdal ® Sleep paralysis Clonazepam, Ritalin ®

A “composition” refers broadly to any composition containing one or more amphetamine prodrugs. The composition can comprise a dry formulation, an aqueous solution, or a sterile composition. Compositions comprising the compounds described herein may be stored in freeze-dried form and may be associated with a stabilizing agent such as a carbohydrate. In use, the composition may be deployed in an aqueous solution containing salts, e.g., NaCl, detergents such as sodium dodecyl sulfate (SDS), and other components.

In one embodiment, the amphetamine prodrug itself exhibits a sustained release profile. Thus, the invention provides a pharmaceutical composition exhibiting a sustained release profile due to the amphetamine prodrug.

In another embodiment, a sustained release profile is enhanced or achieved by including a hydrophilic polymer in the pharmaceutical composition. Suitable hydrophilic polymers include, but are not limited to, natural or partially or totally synthetic hydrophilic gums such as acacia, gum tragacanth, locust bean gum, guar gum, and karaya gum; cellulose derivatives such as methyl cellulose, hydroxymethyl cellulose, hydroxypropylmethyl cellulose, hydroxypropyl cellulose, hydroxyethyl cellulose, and carboxymethyl cellulose; proteinaceous substances such as agar, pectin, carrageen, and alginates; hydrophilic polymers such as carboxypolymethylene; gelatin; casein; zein; bentonite; magnesium aluminum silicate; polysaccharides; modified starch derivatives; and other hydrophilic polymers known in the art. Preferably, the hydrophilic polymer forms a gel that dissolves slowly in aqueous acidic media thereby allowing the amphetamine prodrug to diffuse from the gel in the stomach. Then when the gel reaches the higher pH medium of the intestines, the hydrophilic polymer dissolves in controlled quantities to allow further sustained release. Preferred hydrophilic polymers are hydroxypropyl methylcelluloses such as Methocel ethers, e.g., Methocel E10M® (Dow Chemical Company, Midland, Mich.). One of ordinary skill in the art would recognize a variety of structures, such as bead constructions and coatings, useful for achieving particular release profiles. See, e.g., U.S. Pat. No. 6,913,768.

In addition to the amphetamine prodrug, the pharmaceutical compositions of the invention further comprise one or more pharmaceutical additives. Pharmaceutical additives include a wide range of materials including, but not limited to diluents and bulking substances, binders and adhesives, lubricants, glidants, plasticizers, disintegrants, carrier solvents, buffers, colorants, flavorings, sweeteners, preservatives and stabilizers, and other pharmaceutical additives known in the art. For example, in a preferred embodiment, the pharmaceutical composition comprises magnesium stearate. In another preferred embodiment, the pharmaceutical composition comprises microcrystalline cellulose (e.g., Avicel® PH-102), croscarmellose sodium, and magnesium stearate. See, e.g., Table 62.

Diluents increase the bulk of a dosage form and may make the dosage form easier to handle. Exemplary diluents include, but are not limited to, lactose, dextrose, saccharose, cellulose, starch, and calcium phosphate for solid dosage forms, e.g., tablets and capsules; olive oil and ethyl oleate for soft capsules; water and vegetable oil for liquid dosage forms, e.g., suspensions and emulsions. Additional suitable diluents include, but are not limited to, sucrose, dextrates, dextrin, maltodextrin, microcrystalline cellulose (e.g., Avicel®), microfine cellulose, powdered cellulose, pregelatinized starch (e.g., Starch 1500®), calcium phosphate dihydrate, soy polysaccharide (e.g., Emcosoy®), gelatin, silicon dioxide, calcium sulfate, calcium carbonate, magnesium carbonate, magnesium oxide, sorbitol, mannitol, kaolin, polymethacrylates (e.g., Eudragit®), potassium chloride, sodium chloride, and talc. A preferred diluent is microcrystalline cellulose (e.g., Avicel® PH-102). Preferred ranges for the amount of diluent by weight percent include about 40% to about 90%, about 50% to about 85%, about 55% to about 80%, about 50% to about 60%, and increments therein.

In embodiments where the pharmaceutical composition is compacted into a solid dosage form, e.g., a tablet, a binder can help the ingredients hold together. Binders include, but are not limited to, sugars such as sucrose, lactose, and glucose; corn syrup; soy polysaccharide, gelatin; povidone (e.g., Kollidon®, Plasdone®); Pullulan; cellulose derivatives such as microcrystalline cellulose, hydroxypropylmethyl cellulose (e.g., Methocel®), hydroxypropyl cellulose (e.g., Klucel®), ethylcellulose, hydroxyethyl cellulose, carboxymethylcellulose sodium, and methylcellulose; acrylic and methacrylic acid co-polymers; carbomer (e.g., Carbopol®); polyvinylpolypyrrolidine, polyethylene glycol (Carbowax®); pharmaceutical glaze; alginates such as alginic acid and sodium alginate; gums such as acacia, guar gum, and arabic gums; tragacanth; dextrin and maltodextrin; milk derivatives such as whey; starches such as pregelatinized starch and starch paste; hydrogenated vegetable oil; and magnesium aluminum silicate.

For tablet dosage forms, the pharmaceutical composition is subjected to pressure from a punch and dye. Among other purposes, a lubricant can help prevent the composition from sticking to the punch and dye surfaces. A lubricant can also be used in the coating of a coated dosage form. Lubricants include, but are not limited to, magnesium stearate, calcium stearate, zinc stearate, powdered stearic acid, glyceryl monostearate, glyceryl palmitostearate, glyceryl behenate, silica, magnesium silicate, colloidal silicon dioxide, titanium dioxide, sodium benzoate, sodium lauryl sulfate, sodium stearyl fumarate, hydrogenated vegetable oil, talc, polyethylene glycol, and mineral oil. A preferred lubricant is magnesium stearate. The amount of lubricant by weight percent is preferably less than about 5%, more preferably 4%, 3%, 2%, 1.5%, 1%, or 0.5%, or increments therein.

Glidants can improve the flowability of non-compacted solid dosage forms and can improve the accuracy of dosing. Glidants include, but are not limited to, colloidal silicon dioxide, fumed silicon dioxide, silica gel, talc, magnesium trisilicate, magnesium or calcium stearate, powdered cellulose, starch, and tribasic calcium phosphate.

Plasticizers include both hydrophobic and hydrophilic plasticizers such as, but not limited to, diethyl phthalate, butyl phthalate, diethyl sebacate, dibutyl sebacate, triethyl citrate, acetyltriethyl citrate, acetyltributyl citrate, cronotic acid, propylene glycol, castor oil, triacetin, polyethylene glycol, propylene glycol, glycerin, and sorbitol. Plasticizers are particularly useful for pharmaceutical compositions containing a polymer and in soft capsules and film-coated tablets. In one embodiment, the plasticizer facilitates the release of the amphetamine prodrug from the dosage form.

Disintegrants can increase the dissolution rate of a pharmaceutical composition. Disintegrants include, but are not limited to, alginates such as alginic acid and sodium alginate, carboxymethylcellulose calcium, carboxymethylcellulose sodium (e.g., Ac-Di-Sol®, Primellose®), colloidal silicon dioxide, croscarmellose sodium, crospovidone (e.g., Kollidon®, Polyplasdone®), polyvinylpolypyrrolidine (Plasone-XL®), guar gum, magnesium aluminum silicate, methyl cellulose, microcrystalline cellulose, polacrilin potassium, powdered cellulose, starch, pregelatinized starch, sodium starch glycolate (e.g., Explotab®, Primogel®). Preferred disintegrants include croscarmellose sodium and microcrystalline cellulose (e.g., Avicel® PH-102). Preferred ranges for the amount of disintegrant by weight percent include about 1% to about 10%, about 1% to about 5%, about 2% to about 3%, and increments therein.

In embodiments where the pharmaceutical composition is formulated for a liquid dosage form, the pharmaceutical composition may include one or more solvents. Suitable solvents include, but are not limited to, water; alcohols such as ethanol and isopropyl alcohol; methylene chloride; vegetable oil; polyethylene glycol; propylene glycol; and glycerin.

The pharmaceutical composition can comprise a buffer. Buffers include, but are not limited to, lactic acid, citric acid, acetic acid, sodium lactate, sodium citrate, and sodium acetate.

Any pharmaceutically acceptable colorant can be used to improve appearance or to help identify the pharmaceutical composition. See 21 C.F.R., Part 74. Exemplary colorants include D&C Red No. 28, D&C Yellow No. 10, FD&C Blue No. 1, FD&C Red No. 40, FD&C Green #3, FD&C Yellow No. 6, and edible inks. Preferred colors for gelatin capsules include white, medium orange, and light blue.

Flavorings improve palatability and may be particularly useful for chewable tablet or liquid dosage forms. Flavorings include, but are not limited to maltol, vanillin, ethyl vanillin, menthol, citric acid, fumaric acid, ethyl maltol, and tartaric acid. Sweeteners include, but are not limited to, sorbitol, saccharin, sodium saccharin, sucrose, aspartame, fructose, mannitol, and invert sugar.

The pharmaceutical compositions of the invention can also include one or more preservatives and/or stabilizers to improve storagability. These include, but are not limited to, alcohol, sodium benzoate, butylated hydroxy toluene, butylated hydroxyanisole, and ethylenediamine tetraacetic acid.

Other pharmaceutical additives include gelling agents such as colloidal clays; thickening agents such as gum tragacanth and sodium alginate; wetting agents such as lecithin, polysorbates, and laurylsulphates; humectants; antioxidants such as vitamin E, caronene, and BHT; adsorbents; effervescing agents; emulsifying agents, viscosity enhancing agents; surface active agents such as sodium lauryl sulfate, dioctyl sodium sulfosuccinate, triethanolamine, polyoxyethylene sorbitan, poloxalkol, and quaternary ammonium salts; and other miscellaneous excipients such as lactose, mannitol, glucose, fructose, xylose, galactose, sucrose, maltose, xylitol, sorbitol, chloride, sulfate and phosphate salts of potassium, sodium, and magnesium.

The pharmaceutical compositions can be manufactured according to any method known to those of skill in the art of pharmaceutical manufacture such as, for example, wet granulation, dry granulation, encapsulation, direct compression, slugging, etc. For instance, a pharmaceutical composition can be prepared by mixing the amphetamine prodrug with one or more pharmaceutical additives with an aliquot of liquid, preferably water, to form a wet granulation. The wet granulation can be dried to obtain granules. The resulting granulation can be milled, screened, and blended with various pharmaceutical additives such as water-insoluble polymers and additional hydrophilic polymers. In one embodiment, an amphetamine prodrug is mixed with a hydrophilic polymer and an aliquot of water, then dried to obtain granules of amphetamine prodrug encapsulated by hydrophilic polymer.

After granulation, the pharmaceutical composition is preferably encapsulated, e.g., in a gelatin capsule. The gelatin capsule can contain, for example, kosher gelatin, titanium dioxide, and optional colorants. Alternatively, the pharmaceutical composition can be tableted, e.g., compressed and optionally coated with a protective coating that dissolves or disperses in gastric juices.

The pharmaceutical compositions of the invention can be administered by a variety of dosage forms. Any biologically-acceptable dosage form known in the art, and combinations thereof, are contemplated. Examples of preferred dosage forms include, without limitation, tablets including chewable tablets, film-coated tablets, quick dissolve tablets, effervescent tablets, multi-layer tablets, and bi-layer tablets; caplets; powders including reconstitutable powders; granules; dispersible granules; particles; microparticles; capsules including soft and hard gelatin capsules; lozenges; chewable lozenges; cachets; beads; liquids; solutions; suspensions; emulsions; elixirs; and syrups.

The pharmaceutical composition is preferably administered orally. Oral administration permits the maximum release of amphetamine, provides sustained release of amphetamine, and maintains abuse resistance. Preferably, the amphetamine prodrug releases the amphetamine over a more extended period of time as compared to administering unbound amphetamine.

Oral dosage forms can be presented as discrete units, such as capsules, caplets, or tablets. In a preferred embodiment, the invention provides a solid oral dosage form comprising an amphetamine prodrug that is smaller in size compared to a solid oral dosage form containing a therapeutically equivalent amount of unbound amphetamine. In one embodiment, the oral dosage form comprises a gelatin capsule of size 2, size 3, or smaller (e.g., size 4). The smaller size of the amphetamine prodrug dosage forms promotes ease of swallowing.

Soft gel or soft gelatin capsules may be prepared, for example, by dispersing the formulation in an appropriate vehicle (e.g., vegetable oil) to form a high viscosity mixture. This mixture then is encapsulated with a gelatin based film. The industrial units so formed are then dried to a constant weight.

Chewable tablets can be prepared by mixing the amphetamine prodrug with excipients designed to form a relatively soft, flavored tablet dosage form that is intended to be chewed. Conventional tablet machinery and procedures (e.g., direct compression, granulation, and slugging) can be utilized.

Film-coated tablets can be prepared by coating tablets using techniques such as rotating pan coating methods and air suspension methods to deposit a contiguous film layer on a tablet.

Compressed tablets can be prepared by mixing the amphetamine prodrug with excipients that add binding qualities. The mixture can be directly compressed, or it can be granulated and then compressed.

The pharmaceutical compositions of the invention can alternatively be formulated into a liquid dosage form, such as a solution or suspension in an aqueous or non-aqueous liquid. The liquid dosage form can be an emulsion, such as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion. The oils can be administered by adding the purified and sterilized liquids to a prepared enteral formula, which then is placed in the feeding tube of a patient who is unable to swallow.

For oral administration, fine powders or granules containing diluting, dispersing, and/or surface-active agents can be presented in a draught, in water or a syrup, in capsules or sachets in the dry state, in a non-aqueous suspension wherein suspending agents may be included, or in a suspension in water or a syrup. Liquid dispersions for oral administration can be syrups, emulsions, or suspensions. The syrups, emulsions, or suspensions can contain a carrier, for example, a natural gum, agar, sodium alginate, pectin, methylcellulose, carboxymethylcellulose, saccharose, saccharose with glycerol, mannitol, sorbitol, and polyvinyl alcohol.

The dose range of the amphetamine prodrug for humans will depend on a number of factors including the age, weight, and condition of the patient. Tablets and other dosage forms provided in discrete units can contain a daily dose, or an appropriate fraction thereof, of one or more amphetamine prodrugs. The dosage form can contain a dose of about 2.5 mg to about 500 mg, about 10 mg to about 250 mg, about 10 mg to about 100 mg, about 25 mg to about 75 mg, or increments therein of one or more of the amphetamine prodrugs. In a preferred embodiment, the dosage form contains 30 mg, 50 mg, or 70 mg of an amphetamine prodrug.

The dosage form can utilize any one or any combination of known release profiles including, but not limited to immediate release, extended release, pulse release, variable release, controlled release, timed release, sustained release, delayed release, and long acting.

The pharmaceutical compositions of the invention can be administered in a partial, i.e., fractional dose, one or more times during a 24 hour period. Fractional, single, double, or other multiple doses can be taken simultaneously or at different times during a 24 hour period. The doses can be uneven doses with regard to one another or with regard to the individual components at different administration times. Preferably, a single dose is administered once daily. The dose can be administered in a fed or fasted state.

The dosage units of the pharmaceutical composition can be packaged according to market need, for example, as unit doses, rolls, bulk bottles, blister packs, and so forth. The pharmaceutical package, e.g., blister pack, can further include or be accompanied by indicia allowing individuals to identify the identity of the pharmaceutical composition, the prescribed indication (e.g., ADHD), and/or the time periods (e.g., time of day, day of the week, etc.) for administration. The blister pack or other pharmaceutical package can also include a second pharmaceutical product for combination therapy.

It will be appreciated that the pharmacological activity of the compositions of the invention can be demonstrated using standard pharmacological models that are known in the art. Furthermore, it will be appreciated that the inventive compositions can be incorporated or encapsulated in a suitable polymer matrix or membrane for site-specific delivery, or can be functionalized with specific targeting agents capable of effecting site specific delivery. These techniques, as well as other drug delivery techniques, are well known in the art.

Any feature of the above-describe embodiments can be used in combination with any other feature of the above-described embodiments.

In order to facilitate a more complete understanding of the invention, Examples are provided below. However, the scope of the invention is not limited to specific embodiments disclosed in these Examples, which are for purposes of illustration only.

EXAMPLES

The following abbreviations are used in the Examples and throughout the specification:

-   -   Lys-Amp=L-lysine-d-amphetamine, Lysine-Amphetamine, K-Amp,         K-amphetamine, or 2,6-diaminohexanoic         acid-(1-methyl-2-phenylethyl)-amide, lisdexamphetamine or         Lisdexamfetamine     -   Phe-Amp=Phenylalanine-Amphetamine, F-Amp, or         2-amino-3-phenylpropanoic acid-(1-methyl-2-phenylethyl)-amide     -   Ser-Amp=Serine-Amphetamine, S-Amp, or         2-amino-3-hydroxylpropanoic acid-(1-methyl-2-phenylethyl)-amide,     -   Gly₃-Amp=GGG-Amphetamine, GGG-Amp, or         2-amino-N-({[(1-methyl-2-phenyl-ethylcarbornyl)-methyl]-carbornyl}-methyl)-acetamide     -   BOC=t-butyloxycarbonyl     -   CMC=carboxymethylcellulose     -   DIPEA=di-isopropyl ethyl amine     -   mp=melting point     -   NMR=nuclear magnetic resonance     -   OSu=hydroxysuccinimido ester

Throughout the Examples, unless otherwise specified, doses are described as the amount of d-amphetamine base. Exemplary conversions are provided in Table 2.

TABLE 2 Conversion of d-amphetamine doses (mg) L-lysine-d-amphetamine dimesylate d-amphetamine d-amphetamine sulfate (29.5% d-amphetamine) base (72.8% d-amphetamine) 5.08 1.5 2.06 10.17 3 4.12 20.34 6 8.24 40.68 12 16.48 101.69 30 41.21 203.39 60 82.42 25.00 7.375 10.13 75.00 22.125 30.39 70.00 20.65 28.37 50.00 14.75 20.26 30.00 8.85 12.16

X-Ray Powder Diffraction (XRPD) ANALYSES

XRPD patterns were collected using a PANalytical X'Pert Pro diffractometer. The specimens were analyzed using Cu radiation produced using an Optix long fine-focus source. An alleptically graded multilayer mirror was used to focus the Cu K α X-rays of the source through the specimen and onto the detector. Each specimen was sandwiched between 3 μm thick films, analyzed in transmission geometry, and rotated to optimize orientation statistics. A beam-stop and helium purge was used to minimize the background generated by air scattering. Soller slits were used for the incident and diffracted beams to minimize axial divergence. Diffraction patterns were collected using a scanning position-sensitive detector (X-celerator) located 240 mm from the specimen. The data-acquisition parameters of each diffraction pattern are given in the respective examples, below. Prior to the analysis a silicon specimen (NIST standard reference material 640c) was analyzed to verify the position of the silicon 111 peak.

The XRPD pattern of lisdexamphetamine dimesylate was indexed using proprietary software—PatternMatch 2.4.0, provided by SSCI (West Lafayette, Ind.). The solution was further refined via Pawley refinement using DASH version 3.0, provided by Cambridge Crystallographic Data Centre (Cambridge, UK). The indexed solution was verified and illustrated using CheckCell version Nov. 1, 2004 (ccp14.ac.uk/tutorial/lmgp/index.html).

DSC Analyses

DSC was performed using a TA Instruments differential scanning calorimeter 2920 and Q2000. The respective sample was placed into an aluminum DSC pan, and the weight accurately recorded. The pan was covered with a lid and lift uncrimped. The sample cell was equilibrated at 25° C. and heated under a nitrogen purge at a rate of 10° C./min, up to a final temperature of 250° C. Indium metal was used as the calibration standard. Reported temperatures are at the transition maxima and peak onsets.

FT-Raman Spectroscopy

FT-Raman spectra were acquired on a FT-Raman 960 spectrometer (Thermo Nicolet, Woburn, Mass.). This spectrometer uses an excitation wavelength of 1064 nm. Approximately 0.5 W of Nd:YVO₄ laser power was used to irradiate the respective sample. The Raman spectra were measured with a germanium (Ge) detector. The samples were prepared for analysis by placing the material in a glass tube and positioning the tube in a gold-coated tube holder in the accessory. A total of 256 sample scans were collected from 3600-100 cm⁻¹ at a spectral resolution of 4 cm⁻¹, using Happ-Genzel apodization. Wavelength calibration was performed using sulfur and cyclohexane.

Particle Size Determination

The micrometer bar size was established for both the SEM and light microscope(s) in standard resolution by comparison with a NIST traceable calibration standard. The software for each instrument then determines the size of each pixel in each image, and uses this information for comparison with objects shown in the photomicrograph. The comparison takes into account the magnification of the image and calculates the true size of the object, not the magnified size.

Example 1 General Synthesis of Peptide Amphetamine Conjugates

Peptide conjugates were synthesized by the general method described in FIG. 1. An iterative approach can be used to identify favorable conjugates by synthesizing and testing single amino acid conjugates, and then extending the peptide one amino acid at a time to yield dipeptide and tripeptide conjugates, etc. The parent single amino acid prodrug candidate may exhibit more or less desirable characteristics than its di- or tripeptide offspring candidates. The iterative approach can quickly suggest whether peptide length influences bioavailability.

General Synthesis of Single Amino Acid Amphetamine Conjugates

To a solution of a protected amino acid succinimidyl ester (2.0 eq) in 1,4-dioxane (30 mL) was added d-amphetamine sulfate (1.0 eq) and NMM (4.0 eq). The resulting mixture was allowed to stir for 20 h at 20° C. Water (10 mL) was added, and the solution was stirred for 10 minutes prior to removing solvents under reduced pressure. The crude product was dissolved in EtOAc (100 mL) and washed with 2% AcOH_(aq) (3×100 mL), saturated NaHCO₃ solution (2×50 mL), and brine (1×100 mL). The organic extract was dried over MgSO₄, filtered, and evaporated to dryness to afford the protected amino acid amphetamine conjugate. This intermediate was directly deprotected by adding 4 N HCl in 1,4-dioxane (20 mL). The solution was stirred for 20 h at 25° C. The solvent was evaporated, and the product dried in vacuum to afford the corresponding amino acid amphetamine hydrochloride conjugate. The syntheses of exemplary single amino acid conjugates are depicted in FIG. 2-FIG. 6.

General Synthesis of Dipeptide Amphetamine Conjugates

To a solution of a protected dipeptide succinimidyl ester (1.0 eq) in 1,4-dioxane was added amphetamine sulfate (2.0 eq) and NMM (4.0 eq). The resulting mixture was stirred for 20 h at 25° C. Solvents were removed under reduced pressure. Saturated NaHCO₃ solution (20 mL) was added, and the suspension was stirred for 30 min. IPAC (100 mL) was added, and the organic layer was washed with 2% AcOH_(aq) (3×100 mL) and brine (2×100 mL). The organic extract was dried over Na₂SO₄, and the solvent was evaporated to dryness to yield the protected dipeptide amphetamine conjugate. The protected dipeptide conjugate was directly deprotected by adding 4 N HCl in 1,4-dioxane (20 mL), and the solution stirred for 20 h at 25° C. The solvent was evaporated, and the product was dried in vacuum to afford the corresponding dipeptide amphetamine hydrochloride conjugate.

General Synthesis of Tripeptide Amphetamine Conjugates

An amino acid conjugate was synthesized and deprotected according to the general procedure described above. To a solution of the amino acid amphetamine hydrochloride (1.0 eq) in dioxane (20 mL) was added NMM (5.0 eq) and a protected dipeptide succinate (1.05 eq). The solution was stirred for 20 h at 25° C. The solvent was removed under reduced pressure. Saturated NaHCO₃ solution (20 mL) was added, and the suspension was stirred for 30 min. IPAC (100 mL) was added, and the organic layer was washed with 2% AcOH_(aq) (3×100 mL) and brine (2×100 mL). The organic extract was dried over Na₂SO₄, and the solvent was evaporated to dryness to yield the protected tripeptide amphetamine. Deprotection was directly carried out by adding 4 N HCl in 1,4-dioxane (20 mL). The mixture was stirred for 20 h at 25° C., the solvent was evaporated, and the product was dried in vacuum to afford the respective tripeptide amphetamine hydrochloride conjugate.

The hydrochloride conjugates required no further purification, but many of the deprotected hydrochloride salts were hygroscopic and required special handling during analysis and subsequent in vivo testing.

Example 2 Synthesis of L-lysine-d-amphetamine

L-lysine-d-amphetamine was synthesized by the following methods.

a. Preparation of HCl Salt (see FIG. 3)

i. Coupling

Molar Reagents MW Weight mmoles Equivalents d-amphetamine free base 135.2 4.75 g 35.13 1 Boc-Lys(Boc)-OSu 443.5 15.58 g 35.13 1 Di-iPr-Et-Amine 129 906 mg 7.03 0.2, d = 0.74, 1.22 mL 1,4-Dioxane — 100 mL — —

To a solution of Boc-Lys(Boc)-OSu (15.58 g, 35.13 mmol) in dioxane (100 mL) under an inert atmosphere was added d-amphetamine free base (4.75 g, 35.13 mmol) and DIPEA (0.9 g, 1.22 mL, 7.03 mmol). The resulting mixture was allowed to stir at room temperature overnight. Solvent and excess base were then removed using reduced pressure evaporation. The crude product was dissolved in ethyl acetate and loaded on to a flash column (7 cm wide, filled to 24 cm with silica) and eluted with ethyl acetate. The product was isolated, the solvent reduced by rotary evaporation, and the purified protected amide was dried by high-vac to obtain a white solid. ¹H NMR (DMSO-d₆) δ 1.02-1.11 (m, 2H, Lys γ-CH₂), δ 1.04 (d, 3H, Amp α-CH₃), δ 1.22-1.43 (m, 4H, Lys-β and δ-CH₂), δ 1.37 (18H, Boc, 6×CH₃), δ 2.60-2.72 (2H, Amp CH₂), δ 3.75-3.83, (m, 1H, Lys α-H) δ 3.9-4.1 (m, 1H, Amp α-H), δ 6.54-6.61 (d, 1H, amide NH), δ 6.7-6.77 (m, 1H, amide NH), δ 7.12-7.29 (m, 5H, ArH), δ 7.65-7.71 (m, 1, amide NH); mp=86-88° C.

ii. Deprotection

Molar Reagents MW Weight mmoles Equivalents 4M HCl in dioxane 4 mmol/mL   50 mL 200 6.25 Boc-Lys(Boc)-Amp 463.6 14.84 g 32 1 1,4-Dioxane —   50 mL — —

The protected amide was dissolved in 50 mL of anhydrous dioxane and stirred while 50 mL (200 mmol) of 4M HCl/dioxane was added and stirred at room temperature overnight. The solvents were then reduced by rotary evaporation to afford a viscous oil. Addition of 100 mL MeOH followed by rotary evaporation resulted in a golden colored solid material that was further dried by storage at room temperature under high vacuum. ¹H NMR (DMSO-d₆) δ 0.86-1.16 (m, 2H, Lys γ-CH₂), δ 1.1 (d, 3H, Amp α-CH₃), δ 1.40-1.56 (m, 4H, Lys-β and δ-CH₂), δ 2.54-2.78 (m, 2H, Amp CH₂, 2H, Lys ε-CH₂), 3.63-3.74 (m, 1H, Lys α-H), δ 4.00-4.08 (m, 1H, Amp α-H), δ 7.12-7.31 (m, 5H, Amp ArH), δ 8.13-8.33 (d, 3H, Lys amine) δ 8.70-8.78 (d, 1H, amide NH); mp=120-122° C.

b. Preparation of Mesylate Salt (and See FIG. 2)

Similarly, the mesylate salt of the peptide conjugate can be prepared by using methanesulfonic acid in the deprotection step as described in further detail below.

i. Coupling

A 72-L round-bottom reactor was equipped with a mechanical stirrer, digital thermocouple, and addition funnel and purged with nitrogen. The vessel was charged with Boc-Lys(Boc)-OSu (3.8 kg, 8.568 mol, 1.0 eq) and 1,4-dioxane (20.4 L), and the resulting turbid solution was stirred at 20±5° C. for 10 min. To the mixture was added N-methylmorpholine (950 g, 9.39 mol, 1.09 eq) over a period of 1 min, and the mixture was stirred for 10 min. To the slightly turbid reaction mixture was then added a solution of dextro-amphetamine (1.753 kg, 12.96 mol, 1.51 eq) in 1,4-dioxane (2.9 L) over a period of 30 min, while cooling the reactor externally with an ice/water bath. The internal temperature was kept below 25° C. during the addition. At the end of the addition, a thick white precipitate appeared. The addition funnel was rinsed with 1,4-dioxane (2.9 L) into the reactor, and the reaction mixture was stirred at 22±3° C. TLC monitoring 30 min. after completed addition showed no more remaining Boc-Lys(Boc)-Osu, and the reaction was quenched with DI H₂O (10 L). The mixture was stirred for 1 h at ambient temperature and then concentrated under reduced pressure to afford a dense, white solid.

For the extractions, two solutions were prepared: an acetic acid/salt solution: NaCl (15 kg) and glacial acetic acid (2 kg) in DI H₂O (61 L), and a bicarbonate solution: NaHCO₃ (1.5 kg) in DI H₂O (30 L).

The solid was re-dissolved in IPAC (38 L) and acetic acid/salt solution (39 kg) and transferred into a 150-L reactor. The layers were mixed for 10 min. and then allowed to separate. The organic layer was drained and washed with another portion (39 kg) of acetic acid/salt solution, followed by a wash with bicarbonate solution (31.5 kg). All phase separations occurred within 5 min. To the organic solution was then added silica-gel (3.8 kg; Silica-gel 60). The resulting slurry was stirred for 45 min. and then filtered through filter paper. The filter-cake was washed with IPAC (5×7.6 L). The filtrate and washes were analyzed by TLC, and it was determined that all contained product. The filtrate and washes were combined and concentrated under reduced pressure to afford the crude product as a white solid.

ii. Deprotection

A 45-L carboy was charged with di-Boc-Lys-Amp (3.63 kg, 7.829 mol) and 1,4-dioxane (30.8 L, 8.5 vol), and the mixture was stirred rapidly under nitrogen for 30 min. The resulting solution was filtered, and the filter-cake was rinsed with 1,4-dioxane (2×1.8 L).

The filtrates were then transferred into a 72-L round-bottom flask, which was equipped with a mechanical stirrer, digital thermocouple, nitrogen inlet and outlet, and 5 L addition funnel. The temperature of the reaction mixture was regulated at 21±3° C. with a water bath. To the clear, slightly yellow solution was added methanesulfonic acid (3.762 kg, 39.15 mol, 5 eq) over a period of 1 h while keeping the internal temperature at 21±3° C. Approximately 1 h after completed addition, a white precipitate started to appear. The mixture was stirred at ambient temperature for 20.5 h, after which HPLC monitoring showed the disappearance of all starting material. The mixture was filtered through filter-paper, and the reaction vessel was rinsed with 1,4-dioxane (3.6 L, 1 vol). The filter-cake was washed with dioxane (3×3.6 L) and dried with a rubber dam for 1 h. The material was then transferred to drying trays and dried in a vacuum oven at 55° C. for ˜90 h. This afforded Lys-Amp dimesylate [3.275 kg, 91.8% yield; >99% (AUC)] as a white solid.

Example 3 Synthesis of Ser-Amp

Ser-Amp was synthesized by a similar method (see FIG. 4) except the amino acid starting material was Boc-Ser(O-tBu)-OSu and the deprotection was done using a solution of trifluoroacetic acid instead of HCl.

Example 4 Synthesis of Phe-Amp

Phe-Amp was synthesized by a similar method (see FIG. 5) except the amino acid starting material was Boc-Phe-OSu.

Phe-Amp hydrochloride: hygroscopic; ¹H NMR (400 MHz, DMSO-d₆): δ 8.82 (d, J=8.0 Hz, 1H), 8.34 (bs, 3H), 7.29-7.11 (m, 10H), 3.99 (m, 2H), 2.99 (dd, J=13.6, 6.2 Hz, 1H), 2.88 (dd, J=13.6, 7.2 Hz, 1H), 2.64 (dd, J=13.2, 7.6 Hz, 1H), 2.53 (m, 1H), 1.07 (d, J=6.4 Hz, 3H); ¹³C NMR (100 MHz, DMSO-d₆): δ 167.31, 139.27, 135.49, 130.05, 129.66, 128.78, 128.61, 127.40, 126.60, 53.83, 47.04, 42.15, 37.27, 20.54; HRMS: (ESI) for C₁₈H₂₃N₂O (M+H)⁺: calcd, 283.1810: found, 283.1806.

Example 5 Synthesis of Gly₃-Amp

Gly₃-Amp was synthesized by a similar method (see FIG. 6) except the amino acid starting material was Boc-GGG-OSu.

Gly₃-Amp hydrochloride: mp 212-214° C.; ¹H NMR (400 MHz, DMSO-d₆) δ 7.28 (m, 5H), 3.96 (m, 1H), 3.86 (m, 2H), 3.66 (m, 4H), 2.76 (m, 1H), 2.75 (m, 1H), 1.02 (d, J=6.8 Hz, 3H); ¹³C NMR (100 MHz, DMSO-d₆) δ 168.91, 168.14, 166.85, 139.45, 129.60, 128.60, 126.48, 46.60, 42.27, 20.30. HRMS: (ESI) for C₁₅H₂₂N₄O₃Na (M+Na)⁺: calcd, 329.1590: found, 329.1590.

Example 6 Pharmacokinetics of L-lysine-d-amphetamine diHCl Compared to d-amphetamine Sulfate (ELISA Analysis)

Male Sprague-Dawley rats were provided water ad libitum, fasted overnight, and dosed by oral gavage L-lysine-d-amphetamine diHCl or d-amphetamine sulfate. In all studies, doses contained equivalent amounts of d-amphetamine base. Plasma d-amphetamine concentrations were measured by ELISA (Amphetamine Ultra, 109319, Neogen, Corporation, Lexington, Ky.). The assay is specific for d-amphetamine with only minimal reactivity (0.6%) of the major d-amphetamine metabolite (para-hydroxy-d-amphetamine) occurring. L-lysine-d-amphetamine diHCl was also determined to be essentially unreactive in the ELISA (<1%).

Mean (n=4) plasma concentration curves of d-amphetamine or L-lysine-d-amphetamine diHCl are shown in FIG. 7. Extended release was observed in all four L-lysine-d-amphetamine diHCl dosed animals, and C_(max) was substantially decreased as compared to animals dosed with d-amphetamine sulfate. Plasma d-amphetamine concentrations of individual animals for d-amphetamine or L-lysine-d-amphetamine diHCl are shown in Table 3. The mean plasma d-amphetamine concentrations are shown in Table 4. The time to peak concentration for L-lysine-d-amphetamine diHCl was similar to that of d-amphetamine. Pharmacokinetic parameters for oral administration of d-amphetamine or L-lysine-d-amphetamine diHCl are summarized in Table 5.

TABLE 3 Plasma concentrations of d-amphetamine from individual animals orally administered d-amphetamine or L-lysine-d-amphetamine diHCl (3 mg/kg d- amphetamine base) Time d-amphetamine (ng/ml) L-lysine-d-amphetamine (ng/ml) (hours) Rat #1 Rat #2 Rat #3 Rat #4 Rat #1 Rat #2 Rat #3 Rat #4 0.5 144 157 101 115 52 62 74 44 1 152 78 115 78 48 72 79 57 1.5 85 97 117 95 42 62 76 53 3 34 45 72 38 61 60 71 43 5 20 14 12 15 49 33 44 22 8 3 3 2 2 15 14 12 8

TABLE 4 Mean plasma concentrations of d-amphetamine following oral administration of d-amphetamine or L-lysine-d-amphetamine Plasma d-amphetamine Concentrations (ng/ml) d-amphetamine L-lysine-d-amphetamine Hours Mean ±SD CV Mean ±SD CV 0.5 129 25 20 58 13 22 1 106 35 33 64 14 22 1.5 99 13 14 58 14 25 3 47 17 36 59 11 19 5 15 4 24 37 12 32 8 2 1 35 12 3 24

TABLE 5 Pharmacokinetic parameters of d-amphetamine following oral administration of d-amphetamine or L-lysine-d-amphetamine Mean AUC (0-8 h) Percent C_(max) Percent Peak Percent Drug ng · h/mL Amphetamine (ng/ml) Amphetamine (ng/ml) Amphetamine Amphetamine 341 ± 35 100 111 ± 27 100 129 100 Lys-Amp 333 ± 66 98  61 ± 13 55 64 50

This example illustrates that when lysine is conjugated to the active agent amphetamine, the peak levels of amphetamine are decreased while bioavailability is maintained approximately equal to amphetamine. The bioavailability of amphetamine released from L-lysine-d-amphetamine is similar to that of amphetamine sulfate at the equivalent dose; thus L-lysine-d-amphetamine maintains its therapeutic value. The gradual release of amphetamine from L-lysine-d-amphetamine and decrease in peak levels reduce the possibility of overdose.

Example 7 Oral Bioavailability of L-lysine-d-amphetamine Dimesylate at Various Doses

a. Doses Approximating Therapeutic Human Doses (1.5, 3, and 6 mg/kg)

Mean (n=4) plasma concentration curves of d-amphetamine vs. L-lysine-d-amphetamine are shown for rats orally administered 1.5, 3, and 6 mg/kg in FIG. 8, FIG. 9, and FIG. 10, respectively. Extended release was observed at all three therapeutic doses for L-lysine-d-amphetamine dosed animals. The mean plasma concentrations for 1.5, 3, and 6 mg/kg are shown in Table 6, Table 7, and Table 8, respectively. Pharmacokinetic parameters for oral administration of d-amphetamine vs. L-lysine-d-amphetamine at the various doses are summarized in Table 9.

TABLE 6 Mean plasma concentrations of d-amphetamine vs. L-lysine-d- amphetamine following oral administration (1.5 mg/kg) Plasma Amphetamine Concentrations (ng/ml) d-amphetamine L-lysine-d-amphetamine Hours Mean ±SD CV Mean ±SD CV 0 0 0 0 0 0 0 0.25 103 22 21 31 11 37 0.5 126 20 16 51 23 45 1 101 27 27 68 23 34 1.5 116 28 24 72 10 14 3 66 13 20 91 5 5 5 40 7 18 75 16 22 8 17 2 15 39 13 34

TABLE 7 Mean plasma concentrations of d-amphetamine vs. L-lysine-d- amphetamine following oral administration (3 mg/kg) Plasma Amphetamine Concentrations (ng/ml) d-amphetamine L-lysine-d-amphetamine Hours Mean ±SD CV Mean ±SD CV 0 0 0 0.25 96 41 43 51 49 97 0.5 107 49 46 36 35 96 1 121 17 14 81 44 54 1.5 120 33 27 97 32 33 3 91 30 33 88 13 15 5 62 22 36 91 21 23 8 19 6 33 46 16 34

TABLE 8 Mean plasma concentrations of d-amphetamine vs. L-lysine-d- amphetamine following oral administration (6 mg/kg) Plasma Amphetamine Concentrations (ng/ml) d-amphetamine L-lysine-d-amphetamine Hours Mean ±SD CV Mean ±SD CV 0 0 0 0.25 204 14 7 74 38 51 0.5 186 9 5 106 39 37 1 167 12 7 133 33 24 1.5 161 24 15 152 22 15 3 111 29 26 157 15 10 5 78 9 11 134 18 13 8 35 5 15 79 12 15

TABLE 9 Pharmacokinetic parameters of d-amphetamine following oral administration of d-amphetamine or L-lysine-d-amphetamine 1.5 mg/kg 3 mg/kg 6 mg/kg Parameter Amp K-Amp Amp K-Amp Amp K-Amp AUC 481 538 587 614 807 1005 (ng · h/ml) Percent 100 112 100 105 100 125 C_(max) (ng/ml) 133 93 141 104 205 162 Percent 100 70 100 74 100 79 T_(max) (hours) 0.938 3.5 1 1.56 0.563 2.625 Percent 100 373 100 156 100 466

b. Increased Doses (12, 30, and 60 mg/kg)

Mean (n=4) plasma concentration curves of d-amphetamine vs. L-lysine-d-amphetamine are shown for rats orally administered 12, 30, and 60 mg/kg. At these higher doses, the bioavailability of L-lysine-d-amphetamine was markedly decreased as compared to d-amphetamine.

TABLE 10 Mean plasma concentrations of d-amphetamine vs. L-lysine-d- amphetamine following oral administration (12 mg/kg) Plasma Amphetamine Concentrations (ng/ml) d-amphetamine L-lysine-d-amphetamine Hours Mean ±SD CV Mean ±SD CV 0 NA NA NA NA NA NA 0.25 530 279 53 53 34 64 0.5 621 76 12 99 32 33 1 512 91 18 220 77 35 1.5 519 113 22 224 124 55 3 376 149 40 300 153 51 5 314 123 39 293 153 52 8 103 64 63 211 45 22

TABLE 11 Mean plasma concentrations of d-amphetamine vs. L-lysine-d- amphetamine following oral administration (30 mg/kg) Plasma Amphetamine Concentrations (ng/ml) L-lysine-d- d-amphetamine amphetamine Hours Mean ±SD CV Mean ±SD CV 0 NA NA NA NA NA NA 0.25 2,036 1,262 62 29 16 54 0.5 2,583 1,465 57 88 29 34 1 3,162 772 24 328 30 9 1.5 3,445 191 6 368 99 27 3 2,620 72 3 620 79 13 5 1,535 21 1 730 169 23 8 164 52 32 NA NA NA

TABLE 12 Mean plasma concentrations of d-amphetamine vs. L-lysine-d- amphetamine following oral administration (60 mg/kg) Plasma Amphetamine Concentrations (ng/ml) L-lysine-d- d-amphetamine amphetamine Hours Mean ±SD CV Mean ±SD CV 0 NA NA NA NA NA NA 0.25 3,721 286 8 169 93 55 0.5 3,566 560 16 259 138 53 1 3,556 442 12 420 173 41 1.5 4,142 381 9 506 169 33 3 NA NA NA 686 222 32 5 NA NA NA 612 67 11 8 NA NA NA 870 NA NA

TABLE 13 Pharmacokinetic parameters of d-amphetamine following oral administration of d-amphetamine or L-lysine-d-amphetamine 12 mg/kg 30 mg/kg 60 mg/kg Parameter Amp K-Amp Amp K-Amp Amp K-Amp AUG (ng · h/ml) 2,738 1,958 12,623*   2,387*   5,081**  476** Percent 100 72 100  19 100  9 C_(max) (ng/ml) 621 352 3,726   231 4,101   647 Percent 100 57 100  6 100  16 T_(max) (hours) 0.938 3.5 NA NA NA NA Percent 100 373 NA NA NA NA *0-5 h **0-1.5 h

Example 8 Oral Bioavailability of L-lysine-d-amphetamine Dimesylate at Various Doses Approximating a Range of Therapeutic Human Doses Compared to a Suprapharmacological Dose

Male Sprague-Dawley rats were provided water ad libitum, fasted overnight, and dosed by oral gavage with 1.5, 3, 6, 12, and 60 mg/kg of amphetamine sulfate or L-lysine-d-amphetamine containing the equivalent amounts of d-amphetamine. Concentrations of d-amphetamine were measured by ELISA.

It has been demonstrated that when lysine is conjugated to the active agent d-amphetamine, the levels of d-amphetamine at 30 minutes post-administration are decreased by approximately 50% over a dosage range of 1.5 to 12 mg/kg. However, when a suprapharmacological dose (60 mg/kg) is given, the levels of d-amphetamine from L-lysine-d-amphetamine only reached 8% of those seen for d-amphetamine sulfate (See Table 14, Table 15, and FIG. 15). The substantial decrease in oral bioavailability at a high dose greatly reduces the abuse potential of L-lysine-d-amphetamine.

TABLE 14 Levels of d-amphetamine vs. dosage at 0.5 h post dosing with d-amphetamine sulfate Dose mg/kg 1.5 3 6 12 60 ng/ml 0.5 h 109 ± 59 196 ± 72 294 ± 202 344 ± 126 323 ± 973 Percent 100 100 100 100 100

TABLE 15 Levels of d-amphetamine vs. dosage at 0.5 h post dosing with L-lysine-d-amphetamine Dose mg/kg 1.5 3 6 12 60 ng/ml 0.5 h 45 ± 10 86 ± 26 129 ± 46 172 ± 113 266 ± 18 Percent 41 44 44 50 8

Example 9 Decreased Oral Bioavailability of L-lysine-d-amphetamine Dimesylate at a High Dose

An additional oral PK study illustrated in FIG. 16 shows the d-amphetamine blood levels of a 60 mg/kg dose over an 8 h time course. In the case of d-amphetamine, blood levels quickly reached a very high level, and 8 of 12 animals either died or were sacrificed due to acute symptoms of toxicity. Blood levels (Table 16 and Table 17) of animals administered L-lysine-d-amphetamine, on the other hand, did not peak until 5 hours and reached only a fraction of the levels of the animals receiving amphetamine. (Note: Valid data past 3 h for d-amphetamine could not be determined due to death and sacrifice of animals).

TABLE 16 Mean plasma concentrations of d-amphetamine vs. L-lysine- d-amphetamine following oral administration of a high dose (60 mg/kg) Plasma Amphetamine Concentrations (ng/ml) L-lysine-d- d-amphetamine amphetamine Hours Mean ±SD CV Mean ±SD CV 0 NA NA NA NA NA NA 0.25 2174 907 42 35 17 48 0.5 2643 578 22 81 33 41 1 2828 1319 47 212 30 14 1.5 2973 863 29 200 79 40 3  2944* 95 3 440 133 30 5  153* NA NA 565 100 18 8  1309** NA NA 410 206 50 *n = 2 **n = 1

TABLE 17 Pharmacokinetic parameters of d-amphetamine vs. L-lysine- d-amphetamine Mean AUC Percent C_(max) Percent Peak Percent Drug ng · h/ml d-Amp (ng/ml) d-Amp (ng/ml) d-Amp d- 13420 100 3623 100 2973 100 amphetamine L-lysine-d- 3,143 39 582 16 565 19 amphetamine

Example 10 Oral Bioavailability of d-amphetamine Following Administration of an Extended Release Formulation (Intact or Crushed) or L-lysine-d-amphetamine Dimesylate

Doses of an extended release formulation of d-amphetamine sulfate (Dexedrine Spansule® capsules, GlaxoSmithKline) were orally administered to rats as intact capsules or as crushed capsules and compared to a dose of L-lysine-d-amphetamine containing an equivalent amount of d-amphetamine base (FIG. 20). The crushed capsules showed an increase in C_(max) and AUC_(inf) of 84 and 13 percent, respectively, as compared to intact capsules (Table 18 and Table 19). In contrast, C_(max) and AUC_(inf) of d-amphetamine following administration of L-lysine-d-amphetamine were similar to that of the intact capsule illustrating that extended release is inherent to the compound itself and can not be circumvented by simple manipulation.

TABLE 18 Time-course concentrations of d-amphetamine following oral administration of extended release Dexedrine Spansule ® capsules, crushed extended release Dexedrine Spansule ® capsules, or L-lysine-d-amphetamine (3 mg/kg d- amphetamine base) Plasma Concentration (ng/ml) Crushed L-lysine-d- Hours Intact Spansule ® Capsule Spansule ® Capsule amphetamine 0 0 0 0 0.25 32 46 3 0.5 33 85 5 1 80 147 34 1.5 61 101 60 3 64 66 76 5 46 39 66 8 34 12 38

TABLE 19 Pharmacokinetic parameters of d-amphetamine following oral administration of extended release Dexedrine Spansule ® capsules, crushed extended release Dexedrine Spansule ® capsules, or L-lysine-d-amphetamine (3 mg/kg d-amphetamine base) Intact Crushed L-lysine-d- Parameter Spansule ® Capsule Spansule ® Capsule amphetamine AUC_(0-8 h) 399 449 434 (ng · h/ml) Percent 100 113 109 C_(max) (ng/ml) 80 147 76 Percent 100 184 95 T_(max) (hours) 1 1 3 Percent 100 100 300

This example illustrates the advantage of the invention over conventional controlled release formulations of d-amphetamine.

Example 11 Decreased Intranasal Bioavailability of L-lysine-d-amphetamine Vs. Amphetamine

a. Intranasal (IN) Bioavailability of L-lysine-d-amphetamine Hydrochloride

Male Sprague-Dawley rats were dosed by intranasal administration with 3 mg/kg of amphetamine sulfate or L-lysine-d-amphetamine hydrochloride containing the equivalent amounts of d-amphetamine. L-lysine-d-amphetamine did not release any significant amount of d-amphetamine into circulation by IN administration. Mean (n=4) plasma amphetamine concentration curves of amphetamine vs. L-lysine-d-amphetamine are shown in FIG. 17. Pharmacokinetic parameters for IN administration of L-lysine-d-amphetamine are summarized in Table 20.

TABLE 20 Pharmacokinetic parameters of d-amphetamine vs. L-lysine-d-amphetamine hydrochloride by IN administration AUC (0-1.5 h) Percent C_(max) Percent Drug ng · h/ml d-amphetamine (ng/ml) d-amphetamine Amphetamine 727 100 1,377 100 L-lysine-d- 4 0.5 7 0.5 amphetamine

b. Intranasal Bioavailability of L-lysine-d-amphetamine Dimesylate

The process of part a was repeated using L-lysine-d-amphetamine mesylate salt:

TABLE 21 Pharmacokinetic parameters of d-amphetamine vs. L-lysine-d-amphetamine mesylate salt by IN administration AUC (0-1.0 h) Percent C_(max) Percent Drug ng · h/ml d-amphetamine (ng/ml) d-amphetamine Amphetamine 573 100 1114 100 L-lysine-d- 25 4 26 2 amphetamine mesylate salt

This example illustrates that when lysine is conjugated to the active agent d-amphetamine, the bioavailability by the intranasal route is substantially decreased, thereby diminishing the ability to abuse the drug by this route.

Example 12 Intravenous Bioavailability of Amphetamine Vs. L-lysine-d-amphetamine Dimesylate

Male Sprague-Dawley rats were dosed by intravenous tail vein injection with 1.5 mg/kg of d-amphetamine or L-lysine-d-amphetamine containing the equivalent amount of amphetamine. As observed with IN dosing, the conjugate did not release a significant amount of d-amphetamine. Mean (n=4) plasma concentration curves of amphetamine vs. L-lysine-d-amphetamine are shown in FIG. 19. Pharmacokinetic parameters for IV administration of L-lysine-d-amphetamine are summarized in Table 22.

TABLE 22 Pharmacokinetic parameters of d-amphetamine vs. L-lysine-d-amphetamine by IV administration AUC (0-1.5 h) Percent C_(max) Percent Drug ng · h/ml Amphetamine (ng/ml) Amphetamine Amphetamine 190 100 169 100 K-amphetamine 6 3 5 3

This example illustrates that when lysine is conjugated to the active agent amphetamine, the bioavailability of amphetamine by the intravenous route is substantially decreased, thereby diminishing the ability to abuse the drug by this route.

Example 13 Oral Bioavailability of L-lysine-d-amphetamine Dimesylate Compared to d-Amphetamine at Escalating Doses

The fraction of intact L-lysine-d-amphetamine absorbed following oral administration in rats increased non-linearly in proportion to escalating doses from 1.5 to 12 mg/kg (FIG. 21-FIG. 25). The fraction absorbed at 1.5 mg/kg was only 2.6 percent whereas it increased to 24.6 percent by 12 mg/kg. The fraction absorbed fell to 9.3 percent at the high dose of 60 mg/kg. T_(max) ranged from 0.25 to 3 hours, and peak concentrations occurred earlier for L-lysine-d-amphetamine than for d-amphetamine. L-lysine-d-amphetamine was cleared more rapidly than d-amphetamine with nearly undetectable concentrations by 8 hours at the lowest dose.

The bioavailability (AUC) of d-amphetamine from each drug administered was approximately equivalent at low doses. T_(max) for d-amphetamine from L-lysine-d-amphetamine ranged from 1.5 to 5 hours as compared to 0.5 to 1.5 following administration of d-amphetamine sulfate. The difference in T_(max) was greater at higher doses. C_(max) of d-amphetamine from L-lysine-d-amphetamine was reduced by approximately half as compared to the C_(max) of d-amphetamine from d-amphetamine sulfate administration at doses of 1.5 to 6 mg/kg, doses approximating therapeutic human equivalent doses (HEDs). Thus, at therapeutic doses, the pharmacokinetics of d-amphetamine from L-lysine-d-amphetamine resembled that of a sustained release formulation.

HEDs are defined as the equivalent dose for a 60 kg person in accordance to the body surface area of the animal model. The adjustment factor for rats is 6.2. The HED for a rat dose of 1.5 mg/kg of d-amphetamine, for example, is equivalent to 1.5/6.2×60=14.52 d-amphetamine base; which is equivalent to 14.52/0.7284=19.9 mg d-amphetamine sulfate, when adjusted for the salt content.

TABLE 23 Human Equivalent Doses (HEDs) of d-amphetamine sulfate Rat dose of d-amphetamine Human equivalent dose (HED) of (mg/kg) d-amphetamine sulfate (mg) 1.5 19.9 3 39.9 6 79.7 12 159.4 30 399 60 797.2

At suprapharmacological doses (12 and 60 mg/kg), C_(max) was reduced by 73 and 84 percent, respectively, as compared to d-amphetamine sulfate. For these high doses, the AUCs for d-amphetamine from L-lysine-d-amphetamine were substantially decreased compared to those of d-amphetamine sulfate, with the AUC_(inf) reduced by 76% at the highest dose (60 mg/kg). At 60 mg/kg, the levels of d-amphetamine from d-amphetamine sulfate spiked rapidly; the experimental time course could not be completed due to extreme hyperactivity necessitating humane euthanasia.

In summary, oral bioavailability of d-amphetamine from L-lysine-d-amphetamine decreased to some degree at higher doses. However, pharmacokinetics with respect to dose were nearly linear for L-lysine-d-amphetamine at doses from 1.5 to 60 mg/kg with the fraction absorbed ranging from 52 to 81 percent (extrapolated from 1.5 mg/kg dose). Pharmacokinetics of d-amphetamine sulfate was also nearly linear at lower doses of 1.5 to 6 mg/kg with the fraction absorbed ranging from 62 to 84 percent. In contrast to L-lysine-d-amphetamine, however, parameters were disproportionately increased at higher doses for d-amphetamine sulfate with the fraction absorbed calculated as 101 and 223 percent (extrapolated from 1.5 mg/kg dose), respectively, for the suprapharmacological doses of 12 and 60 mg/kg.

The results suggest that the capacity for clearance of d-amphetamine when delivered as the sulfate salt becomes saturated at the higher doses whereas the gradual hydrolysis of L-lysine-d-amphetamine precludes saturation of d-amphetamine elimination at higher doses. The difference in proportionality of dose to bioavailability (C_(max) and AUC) for d-amphetamine and L-lysine-d-amphetamine is illustrated in FIG. 26-FIG. 28. The pharmacokinetic properties of L-lysine-d-amphetamine as compared to d-amphetamine at the higher doses decrease the ability to escalate doses. This improves the safety and reduces the abuse liability of L-lysine-d-amphetamine as a method of delivering d-amphetamine for the treatment of ADHD or other indicated conditions.

Example 14 Intranasal Bioavailability of L-lysine-d-amphetamine Dimesylate Compared to d-amphetamine

As shown in FIG. 31 and FIG. 32, bioavailability of d-amphetamine following bolus intranasal administration of L-lysine-d-amphetamine was approximately 5 percent of that of the equivalent d-amphetamine sulfate dose with AUC_(inf) values of 56 and 1032, respectively. C_(max) of d-amphetamine following L-lysine-d-amphetamine administration by the intranasal route was also about 5 percent of that of the equivalent amount of d-amphetamine sulfate with values of 78.6 ng/mL and 1962.9 ng/mL, respectively. T_(max) of d-amphetamine concentration was delayed substantially for L-lysine-d-amphetamine (60 minutes) as compared to T_(max) of d-amphetamine sulfate (5 minutes), reflecting the gradual hydrolysis of L-lysine-d-amphetamine. Also, a high concentration of intact L-lysine-d-amphetamine was detected following intranasal administration. These results suggest that intranasal administration of L-lysine-d-amphetamine provides only minimal hydrolysis of L-lysine-d-amphetamine and thus only minimal release of d-amphetamine.

Example 15 Intravenous Bioavailability of L-lysine-d-amphetamine Dimesylate Compared to d-amphetamine

As shown in FIG. 33 and FIG. 34, bioavailability of d-amphetamine following bolus intravenous administration of L-lysine-d-amphetamine was approximately one-half that of the equivalent d-amphetamine sulfate dose with AUC_(inf) values of 237.8 and 420.2, respectively. C_(max) of d-amphetamine following L-lysine-d-amphetamine administration was only about one-fourth that of the equivalent amount of d-amphetamine with values of 99.5 and 420.2, respectively. T_(max) of d-amphetamine concentration was delayed substantially for L-lysine-d-amphetamine (30 minutes) as compared to T_(max) of d-amphetamine sulfate (5 minutes), reflecting the gradual hydrolysis of L-lysine-d-amphetamine. In conclusion, the bioavailability of d-amphetamine by the intravenous route is substantially decreased and delayed when given as L-lysine-d-amphetamine. Moreover, bioavailability is less than that obtained by oral administration of the equivalent dose of L-lysine-d-amphetamine.

Summary of LC/MS/MS Bioavailability Data in Rats

The following tables summarize the bioavailability data collected in the experiments discussed in Examples 13-15. Table 24, Table 25, and Table 26 summarize the pharmacokinetic parameters of d-amphetamine following oral, intranasal, and intravenous administration, respectively, of d-amphetamine or L-lysine-d-amphetamine.

TABLE 24 Pharmacokinetic parameters of d-amphetamine following oral administration of L-lysine-d-amphetamine or d-amphetamine at escalating doses AUC_(inf) Dose C_(max) T_(max) AUC₀₋₈ AUC_(inf) alt. F AUC/Dose C_(max)/Dose Drug (mg/kg) (ng/mL) (h) (ng · h/mL) (ng · h/mL) calc.* (%) (ng · h · kg/mL/mg) (ng · kg/mL/mg) K-Amp 1.5 59.6 3   308 331 376 61 220.7 39.7 Amp 1.5 142.2 0.5   446 461 483 84 307.3 94.8 K-Amp 3 126.9 1.5   721 784 963 72 261.3 42.3 Amp 3 217.2 1.5   885 921 1,059 84 307.0 72.4 K-Amp 6 310.8 3 1,680 1,797 2,009 82 299.5 51.8 Amp 6 815.3 0.25 1,319 1,362 1429 62 227.0 135.9 K-Amp 12 412.6 5 2,426 2,701 2,701 62 225.1 34.4 Amp 12 1,533.1 0.25 4,252 4,428 4,636 101 369.0 127.8 K-Amp 60 2,164.3 5  9995.1 11,478 11,478 52 191.3 36.1 Amp 60 13,735 1  14,281** 48,707 48,707 223 811.8 228.9 *An alternative calculation of AUC_(inf) can be performed using WinNonlin ® software (Version 4.1, Pharsight, Inc., Mountain View, California). **AUC (0-1.5)

TABLE 25 Pharmacokinetic parameters of d-amphetamine following bolus intravenous administration of L-lysine-d-amphetamine (1.5 mg/kg) Dose C_(max) T_(max) AUC₀₋₂₄ AUC₀₋₂₄ AUC_(inf) AUC_(inf) Route Drug (mg/kg) (ng/mL) (h) (ng · h/mL) alt. calc.* (ng · h/mL) alt. calc.* IV K-Amp 1.5 99.5 0.5 237.8 207 237.9 218 IV Amp 1.5 420.2 0.083 546.7 511 546.9 521

TABLE 26 Pharmacokinetic parameters of d-amphetamine following intranasal administration of L-lysine-d-amphetamine Dose C_(max) T_(max) AUC₀₋₁ AUC_(inf) AUC_(inf) Route Drug (mg/kg) (ng/mL) (h) (ng · h/mL) (ng · h/mL) alt. calc.* IN K-Amp 3 78.6 1 56 91 NA IN Amp 3 1962.9 0.083 1032 7291 1,267

Table 27, Table 28, and Table 29 summarize the pharmacokinetic parameters of L-lysine-d-amphetamine following oral, intravenous, and intranasal administration of L-lysine-d-amphetamine.

TABLE 27 Pharmacokinetic parameters of L-lysine-d-amphetamine following oral administration of L-lysine-d-amphetamine at escalating doses Dose C_(max) T_(max) AUC₀₋₈ AUC_(inf) AUC_(inf) F Route Drug (mg/kg) (ng/ml) (ng/ml) (ng · h/mL) (ng · h/mL) alt. calc.* (%) Oral K-Amp 1.5 36.5 0.25 59.4 60 60 2.6 Oral K-Amp 3 135.4 1.5 329.7 332.1 331 7.2 Oral K-Amp 6 676.8 0.25 1156.8 1170.8 1,176 12.8 Oral K-Amp 12 855.9 1 4238.6 4510.4 5,169 24.6 Oral K-Amp 60 1870.3 3 8234.3 8499.9 8,460 9.3

TABLE 28 Pharmacokinetic parameters of L-lysine-d-amphetamine following bolus intravenous administration of L-lysine-d-amphetamine Dose C_(max) T_(max) AUC₀₋₂₄ AUC_(inf) Route Drug (mg/kg) (ng/mL) (h) (ng · h/mL) (ng · h/mL) IV K-Amp 1.5 4513.1 0.083 2,282 2,293

TABLE 29 Pharmacokinetic parameters of L-lysine-d-amphetamine following intranasal administration of L-lysine-d-amphetamine Dose C_(max) T_(max) AUC₀₋₁ AUC_(inf) Route Drug (mg/kg) (ng/mL) (h) (ng · h/mL) (ng · h/mL) IN K-Amp 3 3345.1 0.25 2,580 9,139

Table 30 and Table 31 summarize the percent bioavailability of d-amphetamine following oral, intranasal, and intravenous administration of L-lysine-d-amphetamine as compared to d-amphetamine sulfate.

TABLE 30 Percent bioavailability (AUC_(inf)) of d-amphetamine following administration of L-lysine-d-amphetamine by various routes as compared to bioavailability following administration of d-amphetamine sulfate Dose (mg/kg) d-amphetamine base 1.5 3 6 12 60 HED 19.9 39.9 79.7 159.4 797.2 Oral 72 85 132 61 24 IV 43 NA NA NA NA IN NA 1 NA NA NA

TABLE 31 Percent bioavailability (C_(max)) of d-amphetamine following administration of L-lysine-d-amphetamine by various routes as compared to bioavailability following administration of d-amphetamine sulfate Dose (mg/kg) d-amphetamine base 1.5 3 6 12 60 HED 19.9 39.9 79.7 159.4 797.2 Oral 42 58 38 27 16 IV 24 NA NA NA NA IN NA 4 NA NA NA

Table 32-Table 37 summarize the time-course concentrations of d-amphetamine and L-lysine-d-amphetamine following oral, intranasal, and intravenous administration of d-amphetamine or L-lysine-d-amphetamine.

TABLE 32 Time-course concentrations of d-amphetamine following bolus intravenous administration of L-lysine-d-amphetamine or d-amphetamine sulfate (1.5 mg/kg) Time Concentration (ng/ml) (hours) K-Amp Amp sulfate 0 0 0 0.083 52.8 420.2 0.5 99.5 249.5 1.5 47.1 97.9 3 21.0 38.3 5 9.0 13.2 8 3.7 4.3 24 0.1 0.2

TABLE 33 Time-course concentrations of L-lysine-d-amphetamine following bolus intravenous administration of L-lysine-d-amphetamine (1.5 mg/kg) K-Amp Time concentration (hours) (ng/ml) 0 0 0.083 4513.1 0.5 1038.7 1.5 131.4 3 19.3 5 17.9 8 8.7 24 11.5

TABLE 34 Time-course concentrations of d-amphetamine following oral administration of L-lysine-d-amphetamine at various doses Time Concentration (ng/ml) (hours) 1.5 mg/kg 3 mg/kg 6 mg/kg 12 mg/kg 60 mg/kg 0 0 0 0 0 0 0.25 20.5 25.3 96 54.3 90.9 0.5 34 40.9 140.2 96 175.1 1 46.7 95.1 225.9 233.3 418.8 1.5 40.7 126.9 268.4 266 440.7 3 59.6 105 310.8 356.8 1145.5 5 38.6 107.6 219.5 412.6 2164.3 8 17.1 48 86 225.1 1227.5

TABLE 35 Time-course concentrations of d-amphetamine following oral administration of d-amphetamine sulfate at various doses Time Concentration (ng/ml) (hours) 1.5 mg/kg 3 mg/kg 6 mg/kg 12 mg/kg 60 mg/kg 0 0 0 0 0 0 0.25 107.1 152.6 815.3 1533.1 6243.6 0.5 142.2 198.4 462.7 1216 7931.6 1 105.7 191.3 301.3 828.8 13735.2 1.5 129.5 217.2 314 904.8 11514.9 3 52.6 135.3 134.6 519.9 NA 5 29.5 73.5 77.4 404.3 NA 8 11.5 25.7 31.8 115.4 NA

TABLE 36 Time-course concentrations of d-amphetamine following intranasal administration of L-lysine-d-amphetamine or d-amphetamine sulfate (3 mg/kg) Time Concentration (ng/ml) (hours) K-Amp Amp sulfate 0 0 0 0.083 31.2 1962.9 0.25 45.3 1497.3 0.5 61.3 996.2 1 78.6 404.6 AUC 56 1032.3

TABLE 37 Time-course concentrations of L-lysine-d-amphetamine following intranasal administration of L-lysine-d-amphetamine (3 mg/kg) Concentration Time (ng/ml) (hours) K-Amp 0 0 0.083 3345.1 0.25 3369.7 0.5 2985.8 1 1359.3

Example 16 Bioavailability of L-lysine-d-amphetamine Dimesylate or d-amphetamine Sulfate in Dogs (LC/MS/MS Analysis)

Example Experimental Design:

This was a non-randomized, two-treatment crossover study. All animals were maintained on their normal diet and were fasted overnight prior to each dose administration. L-lysine-d-amphetamine dose was based on the body weight measured on the morning of each dosing day. The actual dose delivered was based on syringe weight before and after dosing. Serial blood samples were obtained from each animal by direct venipuncture of a jugular vein using vacutainer tubes containing sodium heparin as the anticoagulant. Derived plasma samples were stored frozen until shipment to Quest Pharmaceutical Services, Inc. (Newark, Del.). Pharmacokinetic analysis of the plasma assay results was conducted by Calvert. Animals were treated as follows:

Number Dose Dose of Dogs/ Route of Conc. Dose Vol. Level Sex Administration Treatment (mg/mL) (mL/kg) (mg/kg) 3/M PO 1 0.2 10 1 3/M IV 2 1 2 1

Administration of the Test Article:

Oral: The test article was administered to each animal via a single oral gavage. On Day 1, animals received the oral dose by gavage using an esophageal tube attached to a syringe. Dosing tubes were flushed with approximately 20 mL tap water to ensure the required dosing solution was delivered.

Intravenous: On Day 8, animals received L-lysine-d-amphetamine as a single 30-minute intravenous infusion into a cephalic vein.

Sample Collection:

Dosing Formulations: Post-dosing, remaining dosing formulation was saved and stored frozen.

Blood: Serial blood samples (2 mL) were collected using venipuncture tubes containing sodium heparin. Blood samples were taken at 0, 0.25, 0.5, 1, 2, 4, 8, 12, 24, 48, and 72 hours post-oral dosing. Blood samples were collected at 0, 0.167, 0.33, 0.49 (prior to stop of infusion), 0.583, 0.667, 0.75, 1, 2, 3, 4, 8, 12, and 23 hours post-intravenous infusion start. Collected blood samples were chilled immediately.

Plasma: Plasma samples were obtained by centrifugation of blood samples. Duplicate plasma samples (about 0.2 mL each) were transferred into prelabeled plastic vials and stored frozen at approximately −70° C.

Sample Assay:

Plasma samples were analyzed for L-lysine-d-amphetamine and d-amphetamine using a validated LC-MS/MS method with an LLOQ of 1 ng/mL for both analytes.

Microsoft Excel (Version 6, Microsoft Corp., Redmond, Wash.) was used for calculation of mean plasma concentration and graphing of the plasma concentration-time data. Pharmacokinetic analysis (non-compartmental) was performed using the WinNonlin® software program (Version 4.1, Pharsight, Inc. Mountain View, Calif.). The maximum concentration (C_(max)) and the time to C_(max)(T_(max)) were observed values. The area under the plasma concentration-time curve (AUC) was determined using linear-log trapezoidal rules. The apparent terminal rate constant (λz) was derived using linear least-squares regression with visual inspection of the data to determine the appropriate number of points (minimum of 3 data points) for calculating λz. The AUC_(0-inf) was calculated as the sum of AUC_(0-t) and Cpred/λz, where Cpred was the predicted concentration at the time of the last quantifiable concentration. The plasma clearance (CL/F) was determined as the ratio of Dose/AUC_(0-inf). The mean residence time (MRT) was calculated as the ratio of AUMC_(0-inf)/AUC_(0-inf), where AUMC_(0-inf) was the area under the first moment curve from the time zero to infinity. The volume of distribution at steady state (V_(ss)) was estimated as CL*MRT. Half-life was calculated as ln 2/λz. The oral bioavailability (F) was calculated as the ratio of AUC_(0-inf) following oral dosing to AUC_(0-inf) following intravenous dosing. Descriptive statistics (mean and standard deviation) of the pharmacokinetic parameters were calculated using Microsoft Excel.

The objectives of this study were to characterize the pharmacokinetics of L-lysine-d-amphetamine and d-amphetamine following administration of L-lysine-d-amphetamine in male beagle dogs. As shown in FIG. 35, in a cross-over design, L-lysine-d-amphetamine was administered to 3 male beagle dogs orally and intravenously. Blood samples were collected up to 24 and 72 hours after the intravenous and oral doses, respectively.

The mean L-lysine-d-amphetamine and d-amphetamine plasma concentration-time profiles following an intravenous or oral dose of L-lysine-d-amphetamine are presented in FIG. 37 and FIG. 38, respectively. Comparative profiles of L-lysine-d-amphetamine to d-amphetamine following both routes are depicted in FIG. 35 and FIG. 36. Individual plots are depicted in FIG. 39 and FIG. 40. The pharmacokinetic parameters are summarized in Table 38-Table 46. The results indicate that administration of L-lysine-d-amphetamine, as compared to administration of amphetamine, leads to a decrease in patient to patient variability of amphetamine levels (see respective CV values in Table 45).

Following a 30-minute intravenous infusion of L-lysine-d-amphetamine, the plasma concentration reached a peak at the end of the infusion. Post-infusion L-lysine-d-amphetamine concentration declined very rapidly in a biexponential manner, and fell below the quantifiable limit (1 ng/mL) by approximately 8 hours post-dose. Results of non-compartmental pharmacokinetic analysis indicate that L-lysine-d-amphetamine is a high clearance compound with a moderate volume of distribution (V_(ss)) approximating total body water (0.7 L/kg). The mean clearance value was 2087 mL/h·kg (34.8 mL/min·kg) and was similar to the hepatic blood flow in the dog (40 mL/min·kg).

L-lysine-d-amphetamine was rapidly absorbed after oral administration with T_(max) at 0.5 hours in all three dogs. Mean absolute oral bioavailability was 33%, which suggests that L-lysine-d-amphetamine is very well absorbed in the dog. The apparent terminal half-life was 0.39 hours, indicating rapid elimination, as observed following intravenous administration.

Plasma concentration-time profiles of d-amphetamine following intravenous or oral administration of L-lysine-d-amphetamine were similar. See Table 39. At a 1 mg/kg oral dose of L-lysine-d-amphetamine, the mean C_(max) of d-amphetamine was 104.3 ng/mL. The half-life of d-amphetamine was 3.1 to 3.5 hours, much longer when compared to L-lysine-d-amphetamine.

In this study, L-lysine-d-amphetamine was infused over a 30 minute time period. Due to rapid clearance of L-lysine-d-amphetamine it is likely that bioavailability of d-amphetamine from L-lysine-d-amphetamine would decrease if a similar dose were given by intravenous bolus injection. Even when given as an infusion the bioavailability of d-amphetamine from L-lysine-d-amphetamine did not exceed that of a similar dose given orally and the time to peak concentration was substantially delayed. This data further supports that L-lysine-d-amphetamine affords a decrease in the abuse liability of d-amphetamine by intravenous injection.

TABLE 38 Pharmacokinetic parameters of L-lysine-d-amphetamine in male beagle dogs following oral or intravenous administration of L-lysine-d-amphetamine (1 mg/kg d-amphetamine base) Dose C_(max) AUC_(inf) CL/F V_(ss) Route mg/kg ng/mL T_(max) ^(a) h ng · h/mL t_(1/2) h MRT h mL/h · kg mL/kg F % IV 1 1650  0.49 964 0.88 0.33 2087 689 NA (0.00) (178) (0.49-0.49) (97.1) (0.2) (0.03)  (199) (105.9) Oral 1   328.2 0.5  319 0.39 0.81 6351 NA 33 (0.00)   (91.9) (0.5-0.5) (46.3) (0.1) (0.19)   (898.3) (1.9) ^(a)median (range) Abbreviations of pharmacokinetic parameters are as follows: C_(max), maximum observed plasma concentration; T_(max), time when C_(max) observed; AUC_(0-t), total area under the plasma concentration versus time curve from 0 to the last data point; AUC_(0-inf), total area under the plasma concentration versus time curve; t_(1/2), apparent terminal half-life; MRT, mean residence time; CL/F, oral clearance; V_(ss), volume of distribution at steady state; F, bioavailability.

TABLE 39 Pharmacokinetic parameters of d-amphetamine in male beagle dogs following oral or intravenous administration of L-lysine-d- amphetamine (1 mg/kg d-amphetamine base) Dose C_(max) T_(max) ^(a) AUC_(inf) t_(1/2) Route (mg/kg) (ng/mL) (h) (ng · h/mL) (h) IV 1 113.2 1.0 672.5 3.14 (0.00) (3.2) (0.67-2.0)  (85.7) (0.4) Oral 1 104.3 2.0 728.0 3.48 (0.00) (21.8) (2-2) (204.9) (0.4) ^(a)median (range)

TABLE 40 Pharmacokinetics of L-lysine-d-amphetamine in male beagle dogs following 30 min intravenous administration of L-lysine-d-amphetamine (1 mg/kg d- amphetamine base) C_(max) T_(max) ^(a) AUC_(0-t) AUC_(inf) t_(1/2) CL V_(ss) MRT Dog ID (ng/mL) (h) (ng · h/mL) (ng · h/mL) (h) (mL/h/kg) (mL/kg) (h) 1 1470.3 0.49 898.2 900.2 0.72 2222 807.4 0.36 2 1826.4 0.49 1072.3 1076.1 ND^(b) 1859 603.4 0.32 3 1654.2 0.49 914.1 916.9 1.05 2181 656.0 0.30 Mean 1650 0.49 961.5 964.4 0.88 2087 689.0 0.33 SD 178 0.49-0.49 96.0 97.1 0.2  199 105.9 0.03 ^(a)median (range); ^(b)not determined CL, clearance following IV administration

TABLE 41 Pharmacokinetic parameters of L-lysine-d-amphetamine in male beagle dogs following oral administration of L-lysine-d-amphetamine (1 mg/kg d- amphetamine base) C_(max) T_(max) ^(a) AUC_(0-t) AUC_(inf) t_(1/2) CL/F MRT F Dog ID (ng/mL) (h) (ng · h/mL) (ng · h/mL) (h) (mL/h/kg) (h) (%) 1 350.2 0.5 275.3 277.1 0.24 7218 0.68 30.8 2 407.2 0.5 367.8 368.7 0.48 5424 0.74 34.3 3 227.4 0.5 310.8 312.0 0.45 6410 1.03 34.0 Mean 328.2 0.5 318.0 319.3 0.39 6351 0.81 33.0 SD 91.9 0.0 46.7 46.3 0.1 898.3 0.19 1.9 ^(a)median (range)

TABLE 42 Pharmacokinetics of d-amphetamine in male beagle dogs following 30 min. intravenous administration of L-lysine-d-amphetamine (1 mg/kg d-amphetamine base) C_(max) T_(max) ^(a) AUC_(0-t) AUC_(inf) t_(1/2) Dog ID (ng/mL) (h) (ng · h/mL) (ng · h/mL) (h) 1 111.2 2.0 751.9 757.6 3.35 2 116.8 0.67 668.5 673.7 3.43 3 111.4 1.0 557.8 586.1 2.65 Mean 113.2 1.00 659.4 672.5 3.14 SD 3.2 0.67-2.0 97 85.7 0.4 ^(a)median (range)

TABLE 43 Pharmacokinetics of d-amphetamine in male beagle dogs following oral administration of L-lysine-d-amphetamine (1 mg/kg d-amphetamine base) C_(max) T_(max) ^(a) AUC_(0-t) AUC_(inf) t_(1/2) Dog ID (ng/mL) (h) (ng · h/mL) (ng · h/mL) (h) 1 102.1 2.0 686.34 696.89 3.93 2 127.2 2.0 937.57 946.62 3.44 3 83.7 2.0 494.61 540.38 3.06 Mean 104.3 2.0 706.2 728.0 3.48 SD 21.8 2.0-2.0 222.1 204.9 0.4 ^(a)median (range)

TABLE 44 Pharmacokinetics of d-amphetamine in male beagle dogs following oral administration of L-lysine-d-amphetamine or d-amphetamine sulfate (1.8 mg/kg d-amphetamine base) Mean Plasma Standard Coefficient of Time Concentration Deviation (SD) Variation (CV) (hours) Amp K-Amp Amp K-Amp Amp K-Amp 0 0 0 0 0 0 0 1 431.4 223.7 140.7 95.9 32.6 42.9 2 360 291.8 87.6 93.6 24.3 32.1 4 277.7 247.5 68.1 66 24.5 26.7 6 224.1 214.7 59.3 62.1 26.5 28.9 8 175.4 150 66.7 40.1 38.0 26.7 12 81.4 47.6 58.7 19 72.1 39.9 16 33 19.6 28.1 9 85.2 45.9 24 7.2 4.5 4.5 1.7 62.5 37.8

TABLE 45 Pharmacokinetics of d-amphetamine in female beagle dogs following oral administration of L-lysine-d-amphetamine or d-amphetamine sulfate (1.8 mg/kg d-amphetamine base) Mean Plasma Standard Coefficient of Time Concentration Deviation (SD) Variation (CV) (hours) Amp K-Amp Amp K-Amp Amp K-Amp 0 0 0 0 0 0 0 1 217.8 308.8 141.7 40.7 65.1 13.2 2 273.5 308 113.7 29.6 41.6 9.6 4 266 260.9 132.7 37.3 49.9 14.3 6 204.7 212.1 84.5 38.7 41.3 18.2 8 160.1 164.3 72.7 43.5 45.4 26.5 12 79.4 68.7 41.3 31 52.0 45.1 16 25.5 22.3 13.4 4.7 52.5 21.1 24 5.6 5.4 4.1 1.9 73.2 35.2

TABLE 46 Pharmacokinetic parameters of d-amphetamine in male and female beagle dogs following oral administration of L-lysine-d-amphetamine or d-amphetamine sulfate (1.8 mg/kg d-amphetamine base) Males Females Compound Compound Parameter Amp K-Amp Amp K-Amp AUC_(inf) 3088.9 2382.2 2664.5 2569.9 Percent 100 77 100 96 C_(max) 431.4 291.8 308.8 273.5 Percent 100 67 100 89 T_(max) (hours) 1 2 1 2 Percent 100 200 100 200

Example 17 Delayed Cardiovascular Effects of L-lysine-d-amphetamine Dimesylate as Compared to d-amphetamine Following Intravenous Infusion

Systolic and diastolic blood pressure (BP) are increased by d-amphetamine even at therapeutic doses. Since L-lysine-d-amphetamine is expected to release d-amphetamine (albeit slowly) as a result of systemic metabolism, a preliminary study was done using equimolar doses of d-amphetamine or L-lysine-d-amphetamine to 4 dogs (2 male and 2 female). The results suggest that the amide prodrug is inactive and that slow release of some d-amphetamine, occurs beginning 20 minutes after the first dose. Relative to d-amphetamine, however, the effects are less robust. For example, the mean blood pressure is graphed in FIG. 43. Consistent with previously published data (Kohli and Goldberg, 1982), small doses of d-amphetamine were observed to have rapid effects on blood pressure. The lowest dose (0.202 mg/kg, equimolar to 0.5 mg/kg of L-lysine-d-amphetamine) produced an acute doubling of the mean BP followed by a slow recovery over 30 minutes.

By contrast, L-lysine-d-amphetamine produced very little change in mean BP until approximately 30 minutes after injection. At that time, pressure increased by about 20-50%. Continuous release of d-amphetamine is probably responsible for the slow and steady increase in blood pressure over the remaining course of the experiment. Upon subsequent injections, d-amphetamine is seen to repeat its effect in a non-dose dependent fashion. That is, increasing dose 10-fold from the first injection produced a rise to the same maximum pressure. This may reflect the state of catecholamine levels in nerve terminals upon successive stimulation of d-amphetamine bolus injections. Note that the rise in mean blood pressure seen after successive doses of L-lysine-d-amphetamine (FIG. 43) produces a more gradual and less intense effect. Similar results were observed for left ventricular pressure (FIG. 44). These results further substantiate the significant decrease in d-amphetamine bioavailability by the intravenous route when given as L-lysine-d-amphetamine. As a result the rapid onset of the pharmacological effect of d-amphetamine that is sought by persons injecting the drug is eliminated.

TABLE 47 Effects of L-lysine-d-amphetamine on cardiovascular parameters in the anesthetized dog (mean values, n = 2) % % % % Treatment Time SAP Change DAP Change MAP Change LVP Change 0.9% Saline 0 81 0 48 0 61 0 87 0 1 ml/kg 30 87 7 54 11 67 10 87 0 K-Amp 0 84 0 51 0 64 0 86 0 0.5 mg/kg 5 87 4 52 3 66 3 87 2 15 93 11 51 1 67 5 95 11 25 104 25 55 8 73 15 105 22 30 107 28 58 14 77 21 108 26 K-Amp 0 105 0 55 0 74 0 108 0 1.0 mg/kg 5 121 15 63 15 85 15 120 11 15 142 35 73 33 100 35 140 29 25 163 55 97 75 124 68 162 50 30 134 28 73 32 98 32 144 33 K-Amp 0 132 0 71 0 95 0 144 0 5.0 mg/kg 5 142 7 71 0 99 4 151 5 15 176 33 98 39 130 37 184 28 25 126 −5 69 −3 96 1 160 11 30 132 0 70 −1 99 4 163 13 SAP: systolic arterial pressure (mmHg); MAP: mean arterial pressure (mmHg); DAP: diastolic arterial pressure (mmHg); LVP: left ventricular pressure (mmHg); % Change: percent change from respective Time 0.

TABLE 48 Effects of d-amphetamine on cardiovascular parameters in the anesthetized dog (mean values, n = 2) % % % % Treatment Time SAP Change DAP Change MAP Change LVP Change 0.9% Saline 0 110 0 67 0 84 0 105 0 1 ml/kg 30 108 −2 65 −3 82 −2 101 −3 d-amphetamine 0 111 0 67 0 84 0 104 0 0.202 mg/kg 5 218 97 145 117 176 109 214 107 15 168 52 97 45 125 49 157 52 25 148 34 87 30 110 31 142 37 30 140 26 80 20 103 23 135 30 d-amphetamine 0 139 0 78 0 101 0 133 0 0.404 mg/kg 5 240 73 147 88 187 85 238 79 15 193 39 112 44 145 43 191 43 25 166 19 92 17 122 20 168 26 30 160 16 87 11 117 16 163 22 d-amphetamine 0 158 0 87 0 115 0 162 0 2.02 mg/kg 5 228 44 128 48 169 47 227 40 15 196 24 107 23 142 23 200 24 25 189 20 102 17 135 17 192 19 30 183 16 98 13 129 12 187 16

Example 18 Pharmacodynamic (Locomotor) Response to Amphetamine Vs. L-lysine-d-amphetamine diHCl by Oral Administration

Male Sprague-Dawley rats were provided water ad libitum, fasted overnight, and dosed by oral gavage with 6 mg/kg of amphetamine or L-lysine-d-amphetamine containing the equivalent amount of d-amphetamine. Horizontal locomotor activity (HLA) was recorded during the light cycle using photocell activity chambers (San Diego Instruments). Total counts were recorded every 12 minutes for the duration of the test. Rats were monitored in three separate experiments for 5, 8, and 12 hours, respectively. Time vs. HLA counts for d-amphetamine vs. L-lysine-d-amphetamine is shown in FIG. 45 and FIG. 46. In each experiment the time until peak activity was delayed, and the pharmacodynamic effect was evident for an extended period of time for L-lysine-d-amphetamine as compared to d-amphetamine. The total activity counts for HLA of Lys-Amp dosed rats were increased (11-41%) over those induced by d-amphetamine in all three experiments.

TABLE 49 Locomotor activity of rats orally administered d-amphetamine vs. L-lysine-d-amphetamine (5 h) Peak of Time of Time of Total Total Activity activity Peak Last Count Test Activity Counts Above (Counts (Counts Above 200 Material Counts Baseline per 0.2 h) per 0.2 h) per 0.2 h Vehicle 4689 4174 80 1.4 — K-Amp 6417 5902 318 1.8   5 h Amp 515 0 291 0.6 2.6 h

TABLE 50 Locomotor activity of rats orally administered d-amphetamine vs. L-lysine-d-amphetamine (12 h) Peak of Time of Time of Total Total Activity activity Peak Last Count Test Activity Counts Above (Counts (Counts Above 200 Material Counts Baseline per 0.2 h) per 0.2 h) per 0.2 h Vehicle 936 0 81 7.2 — K-Amp 8423 7487 256 1.8 8.6 h Amp 6622 5686 223 0.6 6.4 h

Example 19 Pharmacodynamic Response to d-amphetamine Vs. L-lysine-d-amphetamine diHCl by Intranasal Administration

Male Sprague-Dawley rats were dosed by intranasal administration with d-amphetamine or L-lysine-d-amphetamine (1.0 mg/kg). In a second set of similarly dosed animals, carboxymethyl cellulose (CMC) was added to the drug solutions at a concentration of 62.6 mg/ml (approximately 2-fold higher than the concentration of L-lysine-d-amphetamine and 5-fold higher than the d-amphetamine content). The CMC drug mixtures were suspended thoroughly before each dose was delivered. Locomotor activity was monitored using the procedure described in Example 18. As shown in FIG. 47 and FIG. 48, the activity vs. time (1 hour or 2 hours) is shown for amphetamine/CMC vs. L-lysine-d-amphetamine and compared to that of amphetamine vs. L-lysine-d-amphetamine CMC. As seen in FIG. 47, addition of CMC to L-lysine-d-amphetamine decreased the activity response of IN dosed rats to levels similar to the water/CMC control, whereas no effect was seen on amphetamine activity by the addition of CMC. The increase in activity over baseline of L-lysine-d-amphetamine with CMC was only 9% compared to 34% for L-lysine-d-amphetamine without CMC when compared to activity observed for d-amphetamine dosed animals (Table 51). CMC had no observable effect on d-amphetamine activity induced by IN administration.

TABLE 51 Locomotor activity of intranasal d-amphetamine vs. L-lysine-d-amphetamine with and without CMC Total Activity Total Activity Counts Percent Drug n Counts (1 h) Above Baseline Amp Amp 3 858 686 100 Amp CMC 3 829 657 100 K-Amp 4 408 237 35 K-Amp CMC 4 232 60 9 Water 1 172 0 0 Water CMC 1 172 0 0

Example 20 Pharmacodynamic Response to d-amphetamine Vs. L-lysine-d-amphetamine diHCl by Intravenous Administration

Male Sprague-Dawley rats were dosed by intravenous administration with d-amphetamine or L-lysine-d-amphetamine (1.0 mg/kg). The activity expressed as total activity counts over a three hour period of time is shown in FIG. 49. The activity induced by L-lysine-d-amphetamine was substantially decreased, and time to peak activity was delayed. The increase in activity over baseline of L-lysine-d-amphetamine was 34% for L-lysine-d-amphetamine when compared to activity observed for d-amphetamine dosed animals (Table 52).

TABLE 52 Total activity counts after intravenous administration of d-amphetamine vs. L-lysine-d-amphetamine Total Activity Percent Drug n Counts (3 h) Above Baseline Amp Amp 3 1659 1355 100 K-Amp 4 767 463 34 Water 1 304 0 0

Example 21 Decrease in Toxicity of Orally Administered L-lysine-d-amphetamine diHCl

Three male and three female Sprague Dawley rats per group were given a single oral administration of L-lysine-d-amphetamine at 0.1, 1.0, 10, 60, 100, or 1000 mg/kg (Table 53). Each animal was observed for signs of toxicity and death on Days 1-7 (with Day 1 being the day of the dose), and one rat/sex/group was necropsied upon death (scheduled or unscheduled).

TABLE 53 Dosing chart for oral administration of L-lysine-d-amphetamine toxicity testing No. of Animals Dose Concentration Groups M F Test Article (mg/kg) (mg/mL) 1 3 3 L-lysine-d-amphetamine 0.1 0.01 2 3 3 L-lysine-d-amphetamine 1.0 0.1 3 3 3 L-lysine-d-amphetamine 10 1.0 4 3 3 L-lysine-d-amphetamine 60 6.0 5 3 3 L-lysine-d-amphetamine 100 10 6 3 3 L-lysine-d-amphetamine 1000 100

Key observations of this study include:

-   -   All animals in Groups 1-3 showed no observable signs throughout         the conduct of the study.     -   All animals in Groups 4-6 exhibited increased motor activity         within two hours post-dose and which lasted into Day 2.     -   One female rat dosed at 1000 mg/kg was found dead on Day 2.         Necropsy revealed chromodacryorrhea, chromorhinorrhea, distended         stomach (gas), enlarged adrenal glands, and edematous and         distended intestines.     -   A total of 4 rats had skin lesions of varying degrees of         severity on Day 3.     -   One male rat dosed at 1000 mg/kg was euthanatized on Day 3 due         to open skin lesions on the ventral neck.     -   All remaining animals appeared normal from Day 4 through Day 7.

Animals were observed for signs of toxicity at 1, 2, and 4 h post-dose, and once daily for 7 days after dosing and cage-side observations were recorded. Animals found dead, or sacrificed moribund were necropsied and discarded.

Cage-side observations and gross necropsy findings are summarized above. The oral LD50 of d-amphetamine sulfate is 96.8 mg/kg. For L-lysine-d-amphetamine dimesylate, although the data are not sufficient to establish a lethal dose, the study indicates that the lethal oral dose of L-lysine-d-amphetamine is above 1000 mg/kg because only one death occurred out of a group of six animals. Although a second animal in this dose group was euthanatized on Day 3, it was done for humane reasons and it was felt that this animal would have fully recovered. Observations suggested drug-induced stress in Groups 4-6 that is characteristic of amphetamine toxicity (NTP, 1990; NIOSH REGISTRY NUMBER: SI1750000; Goodman et. al., 1985). All animals showed no abnormal signs on Days 4-7 suggesting full recovery at each treatment level.

The lack of data to support an established lethal dose is believed to be due to a putative protective effect of conjugating amphetamine with lysine. Intact L-lysine-d-amphetamine has been shown to be inactive, but becomes active upon metabolism into the unconjugated form (d-amphetamine). Thus, at high doses, saturation of metabolism of L-lysine-d-amphetamine into the unconjugated form may explain the lack of observed toxicity, which was expected at doses greater than 100 mg/kg, which is consistent with d-amphetamine sulfate (NTP, 1990). The formation rate of d-amphetamine and the extent of the formation of amphetamine may both attribute to the reduced toxicity. Alternatively, oral absorption of L-lysine-d-amphetamine may also be saturated at such high concentrations, which may suggest low toxicity due to limited bioavailability of L-lysine-d-amphetamine.

Example 22 In Vitro Assessment of L-lysine-d-amphetamine diHCl Pharmacodynamic Activity

It was anticipated that the acylation of amphetamine, as in the amino acid conjugates discussed here, would significantly reduce the stimulant activity of the parent drug. For example, Marvola (1976) showed that N-acetylation of amphetamine completely abolished the locomotor activity increasing effects in mice. To confirm that the conjugate was not directly acting as a stimulant, we tested (NovaScreen, Hanover, Md.) the specific binding of Lys-Amp (10⁻⁹ to 10⁻⁵ M) to human recombinant dopamine and norepinephrine transport binding sites using standard radioligand binding assays. The results (Table 54) indicate that the Lys-Amp did not bind to these sites. It seems unlikely that the conjugate retains stimulant activity in light of these results. (Marvola M. (1976) “Effect of acetylated derivatives of some sympathomimetic amines on the acute toxicity, locomotor activity and barbiturate anesthesia time in mice.” Acta Pharmacol Toxicol (Copenh) 38(5): 474-89).

TABLE 54 Results from radioligand binding experiments with L-lysine-d-amphetamine Reference Ki (M) for Assay Radioligand Compound Ref. Cpd. Activity* NE Transporter [3H]-Nisoxetine Desipramine 4.1 × 10⁻⁹ No DA Transporter [3H]-WIN35428 GBR-12909 7.7 × 10⁻⁹ No *No activity is defined as producing between −20% and 20% inhibition of radioligand binding (Novascreen).

TABLE 55 Percent inhibition of DAT and NET with L-lysine-d-amphetamine L-lysine-d-amphetamine (mol/L) % inhibition DAT % inhibition NET 10⁻⁹ −10.46 8.15 10⁻⁷ 11.52 −11.75 10⁻⁵ −0.71 13.89

Example 23 In Vitro Assessment to Release Amphetamine from L-lysine-d-amphetamine Dimesylate

“Kitchen tests” were performed in anticipation of attempts by illicit chemists to release free amphetamine from the amphetamine conjugate. Preferred amphetamine conjugates are resistant to such attempts. Initial kitchen tests assessed the amphetamine conjugates' resistance to water, acid (vinegar), and base (baking powder and baking soda) where in each case, the sample was heated to boiling for 20-60 minutes. L-lysine-d-amphetamine and GGG-Amp released no detectable free amphetamine.

TABLE 56 In vitro assessment Vinegar Tap Water Baking Powder Baking Soda L-lysine-d- 0% 0% 0% 0% amphetamine Gly₃-Amp 0% 0% 0% 0%

Amphetamine conjugate stability was assessed under concentrated conditions, including concentrated HCl and in 10 N NaOH solution at elevated temperatures. Lys-Amp stock solutions were prepared in H₂O and diluted 10-fold with concentrated HCl to a final concentration of 0.4 mg/mL and a final volume of 1.5 mL. Samples were heated in a water bath to about 90° C. for 1 hour, cooled to 20° C., neutralized, and analyzed by HPLC for free d-amphetamine. The results suggest that only a minimal amount of d-amphetamine is released under these concentrated conditions.

TABLE 57 Stability under concentrated conditions % AUC solution Lys-Amp d-amphetamine 10 N NaOH 99 <1 conc. HCl 96 4

Amphetamine conjugate stability was assessed under acidic conditions.

TABLE 58 Acids used for stability study Acid Concentrations Hydrochloric acid 10%, 25%, 50%, 75%, and concentrated Acetic acid 10%, 25%, 50%, 75%, and concentrated Sulfuric acid 10%, 25%, 50%, 75%, and concentrated Phosphoric acid 10%, 25%, 50%, 75%, and concentrated Nitric acid 10%, 25%, 50%, 75%, and concentrated Citric acid 10%, 25%, 50%, 75%, and saturated

At ambient temperature, only a limited amount of d-amphetamine was released. At 90° C., only a limited amount of d-amphetamine was released, but the decomposition of L-lysine-d-amphetamine was more pronounced. This suggested that the amide bond is stable, and that the conjugate usually degrades before an appreciable amount is hydrolyzed. At reflux conditions, concentrated hydrochloric acid and 50% sulfuric acid released 85% and 59%, respectively, of the d-amphetamine content, but rendered the drug in undesirable acidic solution. The process for recovering d-amphetamine from the acidic solution further reduces the yield.

In a similar test, reflux in concentrated HCl resulted in some hydrolysis after 5 hours (28%) with further hydrolysis occurring after 22 hours (76%). Reflux in concentrated H₂SO₄ for 2 hours resulted in complete decomposition of Lys-Amp and potentially released d-amphetamine. As described above, recovery of d-amphetamine from the acidic solution would further reduce the yield.

Amphetamine conjugate stability was also assessed under basic conditions, including variable concentrations of sodium hydroxide, potassium hydroxide, sodium carbonate, ammonium hydroxide, diethyl amine, and triethyl amine. The maximum d-amphetamine release was 25.4% obtained by 3M sodium hydroxide; all other basic conditions resulted in a release of less than 3%.

Example 24 Stability of L-lysine-d-amphetamine Dimesylate Under Treatment with Commercially Available Products

The stability of L-lysine-d-amphetamine dimesylate was assessed under treatment commercially available acids, bases, and enzyme cocktails. For acids and bases (Table 59), 10 mg of Lys-Amp was mixed with 2 mL of each stock solution, and the solution was shaken at 20° C. For enzyme treatment (Table 60), 10 mg Lys-Amp was mixed with 5 mL of each enzyme cocktail, and the solution was shaken at 37° C. Each aliquot (0, 1, and 24 h) was neutralized and filtered prior to analysis by HPLC. Many of the commercially available reagents also contained various solvents and/or surfactants.

Unless otherwise indicated, solutions were used directly from the container and were combined with neat Lys-Amp solid. Lewis Red Devil® Lye, Enforcer Drain Care® Septic Treatment, and Rid-X® Septic Treatment were prepared as saturated solutions in H₂O. Enzymes used were purchased from Sigma and directly dissolved in water (3 mg/mL pepsin, 10 mg/mL pancreatin, 3 mg/mL pronase, 3 mg/mL esterase), while enzyme-containing nutraceuticals such as Omnigest® and VitälZym® were first either crushed or opened (1 tablet or capsule per 5 mL of H₂O).

The commercial acids and bases were ineffective in hydrolyzing Lys-Amp. Only treatment with Miracle-Gro® (7% release) and Olympic® Deck Cleaner (4% release) showed any release, but even after 24 hours, the amount of d-amphetamine was negligible. Among the enzyme products, only pure esterase (19% release) or pronase (24% release) mixtures successfully cleaved lysine (after 24 hours).

TABLE 59 Stability of L-lysine-d-amphetamine dimesylate under treatment with commercially available acids and bases d-amphetamine (1) (w.t. % d-amphetamine) Solution (active ingredients) 1 h 24 h Lysol ® Toilet (HCl) 0 0 Crete-nu (75% H₃PO₄) 0 0 CLR ® (sulfamic acid, hydroxyacetic acid) 0 0 Roebic ® Drain Flow (90% H₂SO₄) 0 0 Crown ® Muriatic Acid (31.45% HCl) 0 0 Liquid-Plumr ® (NaOH, NaClO, H₂O₂) 0 0 Brasso ® (NH₄OH) 0 0 Johnson ® Wax Degreaser (K₂CO₃) 0 0 Miracle-Gro ® (Urea, K₃PO₄) 0 7 Lewis Red Devil ® Lye (NaOH) 0 0 Drain Power (NaOH, NaClO) 0 0 Savogran TSP (Na₃PO₄) 0 0 Johnson ® Wax Stripper (NaOH) 0 0 Olympic ® Deck Cleaner (NaOH, NaClO) 0 4 Windex ® (NH₄OH) 0 0 Greased Lightning ® (basic components) 0 0

TABLE 60 Stability of L-lysine-d-amphetamine dimesylate under treatment with commercially available enzyme cocktails d-amphetamine (1) (w.t. % d-amphetamine) Solution 1 h 24 h Cellfood ® 0 0 Drano ® Max with Bacteria 0 0 VitälZym ® 0 0 Omnigest ® 0 0 Enforcer ® Septic 0 0 Rid-X ® Septic 0 0 Esterase 0 19 Pancreatin 0 0 Pepsin 0 0 Pronase 0 24

Example 25 Bioavailability of Various Peptide Amphetamine Conjugates (HCl Salts) Administered by Oral, Intranasal, and Intravenous Routes

Oral administration: Male Sprague-Dawley rats were provided water ad libitum, fasted overnight, and dosed by oral gavage with amphetamine or amino acid-amphetamine conjugates containing the equivalent amount of amphetamine.

Intranasal administration: Male Sprague-Dawley rats were dosed by intranasal administration with amphetamine or lysine-amphetamine (1.8 mg/kg).

The relative in vivo performance of various amino acid-amphetamine compounds is shown in FIG. 50-FIG. 58 and summarized in Table 61. Intranasal bioavailability of amphetamine from Ser-Amp was decreased to some degree relative to free amphetamine. However, this compound was not bioequivalent with amphetamine by the oral route of administration. Phenylalanine was bioequivalent with amphetamine by the oral route of administration, however, little or no decrease in bioavailability by parenteral routes of administration was observed. Gly₃-Amp had nearly equal bioavailability (90%) by the oral route accompanied by a decrease in C_(max) (74%). Additionally, Gly₃-Amp showed a decrease in bioavailability relative to amphetamine by intranasal and intravenous routes.

TABLE 61 Percent bioavailability of amino acid amphetamine compounds administered by oral, intranasal, or intravenous routes Oral Intranasal Intravenous Percent Percent Percent Percent Percent Percent Drug AUC C_(max) AUC C_(max) AUC C_(max) Amphetamine 100 100 100 100 100 100 E-Amp 73 95 NA NA NA NA EE-Amp 26 74 NA NA NA NA EEE-Amp 69 53 10 10 NA NA L-Amp 65 81 NA NA NA NA S-Amp 79/55 62/75 76 65 NA NA G-Amp 81 78 65 53 NA NA GG-Amp 79 88 88 85 NA NA GGG-Amp 111/68  74/73 32 38 45 46 F-Amp 95 91 97 95 87 89 EEF-Amp 42 73 39 29 NA NA FF-Amp 27 64 NA NA NA NA Gulonate-Amp 1 1 0.4 0.5 3 5 K-Amp 98 55 0.5 0.5 3 3 KG-Amp 69 71 13 12 NA NA dKlK-Amp 16 7 2 2 NA NA LE-Amp 40 28 6 6 NA NA H-Amp 16 21 22 42 NA NA P-Amp 6 3 2 2 NA NA PP-Amp 61 80 47 43 NA NA Y-Amp 25 20 21 20 NA NA I-Amp 71 52 73 97 NA NA

Several single amino acid amphetamine conjugates had comparable oral bioavailability (80-100%) to d-amphetamine. Lys, Gly, and Phe conjugates, for example, all demonstrated similar oral bioavailability to the parent drug. Dipeptide prodrugs generally showed lower bioavailability than the respective amino acid analogs, and tripeptide compounds displayed no discernable trend. Several amino acid amphetamine conjugates had decreased parenteral bioavailability. Preferred conjugates, such as Lys-Amp, exhibit both oral bioavailability comparable to d-amphetamine and decreased parenteral bioavailability compared to d-amphetamine.

Example 26 Decreased Oral C_(max) of d-amphetamine Conjugates

Male Sprague-Dawley rats were provided water ad libitum, fasted overnight, and dosed by oral gavage with amphetamine conjugate or d-amphetamine sulfate. All doses contained equivalent amounts of d-amphetamine base. Plasma d-amphetamine concentrations were measured by ELISA (Amphetamine Ultra, 109319, Neogen, Corporation, Lexington, Ky.). The assay is specific for d-amphetamine with only minimal reactivity (0.6%) of the major d-amphetamine metabolite (para-hydroxy-d-amphetamine) occurring. Plasma d-amphetamine and L-lysine-d-amphetamine concentrations were measured by LC/MS/MS where indicated in examples.

Example 27 Decreased Intranasal Bioavailability (AUC and C_(max)) of d-amphetamine Conjugates

Male Sprague-Dawley rats were provided water ad libitum and doses were administered by placing 0.02 ml of water containing amphetamine conjugate or d-amphetamine sulfate into the nasal flares. All doses contained equivalent amounts of d-amphetamine base. Plasma d-amphetamine concentrations were measured by ELISA (Amphetamine Ultra, 109319, Neogen, Corporation, Lexington, Ky.). The assay is specific for d-amphetamine with only minimal reactivity (0.6%) of the major d-amphetamine metabolite (para-hydroxy-d-amphetamine) occurring. Plasma d-amphetamine and L-lysine-d-amphetamine concentrations were measured by LC/MS/MS where indicated in examples.

Example 28 Decreased Intravenous Bioavailability (AUC and C_(max)) of d-amphetamine Conjugates

Male Sprague-Dawley rats were provided water ad libitum, and doses were administered by intravenous tail vein injection of 0.1 ml of water containing amphetamine conjugate or d-amphetamine sulfate. All doses contained equivalent amounts of d-amphetamine base. Plasma d-amphetamine concentrations were measured by ELISA (Amphetamine Ultra, 109319, Neogen, Corporation, Lexington, Ky.). The assay is specific for d-amphetamine with only minimal reactivity (0.6%) of the major d-amphetamine metabolite (para-hydroxy-d-amphetamine) occurring. Plasma d-amphetamine and L-lysine-d-amphetamine concentrations were measured by LC/MS/MS where indicated in examples.

Example 29 Attachment of Amphetamine to Variety of Chemical Moieties

The above examples demonstrate the use of an amphetamine conjugated to a chemical moiety, such as an amino acid, which is useful in reducing the potential for overdose while maintaining its therapeutic value. The effectiveness of binding amphetamine to a chemical moiety was demonstrated through the attachment of amphetamine to lysine (K), however, the above examples are meant to be illustrative only. The attachment of amphetamine to any variety of chemical moieties (i.e., peptides, glycopeptides, carbohydrates, nucleosides, or vitamins) as described below through similar procedures using the following exemplary starting materials.

Amphetamine Synthetic Examples Synthesis of Gly₂-Amp

-   -   Gly₂-Amp was synthesized by a similar method except the amino         acid starting material was Boc-Gly-Gly-OSu.

Synthesis of Glu₂-Phe-Amp

-   -   Glu2-Phe-Amp was synthesized by a similar method except the         amino acid starting material was Boc-Glu(OtBu)-Glu(OtBu)-OSu and         the starting drug conjugate was Phe-Amp (see Phe-Amp synthesis).

Synthesis of His-Amp

His-Amp was synthesized by a similar method except the amino acid starting material was Boc-His(Trt)-OSu.

Synthesis of Lys-Gly-Amp

Lys-Gly-Amp was synthesized by a similar method except the amino acid starting material was Boc-Lys(Boc)-OSu and the starting drug conjugate was Gly-Amp (see Gly-Amp synthesis).

Synthesis of Lys-Glu-Amp

Lys-Glu-Amp was synthesized by a similar method except the amino acid starting material was Boc-Lys(Boc)-OSu and the starting drug conjugate was Glu-Amp.

Synthesis of Glu-Amp

Glu-Amp was synthesized by a similar method except the amino acid starting material was Boc-Glu(OtBu)-OSu.

Synthesis of (d)-Lys-(l)-Lys-Amp

(d)-Lys-(l)-Lys-Amp was synthesized by a similar method except the amino acid starting material was Boc-(d)-Lys(Boc)-(l)-Lys(Boc)-OSu.

Synthesis of Gulonic acid-Amp

Gul-Amp was synthesized by a similar method except the carbohydrate starting material was gulonic acid-OSu.

Example 30 Lack of Detection of L-lysine-d-amphetamine diHCl in Brain Tissue Following Oral Administration

Male Sprague-Dawley rats were provided water ad libitum, fasted overnight, and dosed by oral gavage with L-lysine-d-amphetamine or d-amphetamine sulfate. All doses contained equivalent amounts of d-amphetamine base. As shown in FIG. 59, similar levels of d-amphetamine were detected in serum as well as in brain tissue following administration of d-amphetamine sulfate or L-lysine-d-amphetamine. The d-amphetamine from L-lysine-d-amphetamine showed a sustained presence in the brain as compared to levels of d-amphetamine from d-amphetamine sulfate. The conjugate L-lysine-d-amphetamine was present in appreciable amounts in serum but was not detected in brain tissue indicating that the conjugate does not cross the blood brain barrier to access the central nervous system site of action.

Example 31 Pharmaceutical Composition of L-lysine-d-amphetamine Dimesylate

A gelatin capsule dosage form was prepared in three dosage strengths. The hard gelatin capsules were printed with NRP104 and the dosage strength. The capsule fill contains a white to off-white finely divided powder uniform in appearance.

TABLE 62 Composition of L-lysine-d-amphetamine dimesylate capsules Quantity (mg) Ingredient 30 50 70 Placebo Function Grade L-lysine-d- 30.0 50.0 70.0 0.0 Active amphetamine dimesylate Microcrystalline 151 70.0 98.0 144.0 Filler/ NF (Avicel ® Cellulose diluent, PH-102) disintegrant Croscarmellose 4.69 3.12 4.37 3.75 Disintegrant NF Sodium Magnesium 1.88 1.88 2.63 2.25 Lubricant NF (5712) Stearate Gelatin Capsule White/ White/ Med. White/ Carrier NF Size 3 Med. Lt. Orange/ White Orange Blue Lt. Blue Total 187.5 125 175 150

Other diluents, disintegrants, lubricants, and colorants, etc. may be used. Also, a particular ingredient can be used to serve a different function than those listed above.

The pharmaceutical composition was prepared by milling de-lumped L-lysine-d-amphetamine dimesylate (size 20 mesh) with microcrystalline cellulose. The mixture was sieved through a 30 mesh screen and then mixed with croscarmellose sodium. Pre-screened magnesium stearate (size 30 mesh) was added, and the composition was mixed until uniform to form the capsule fill.

Example 32 Clinical Pharmacokinetic Evaluation and Oral Bioavailability of L-lysine-d-amphetamine Dimesylate 70 mg Capsules Administered to Healthy Adults Under Fasting Conditions for 7 Days

In this open-label, single-arm study, healthy adults between the ages of 18 to 55 years were administered 70 mg of L-lysine-d-amphetamine dimesylate with 8 ounces of water once daily (7 am) for 7 consecutive days. Patients fasted for at least 10 hours before and 4 hours after final dosing. Venous blood samples (7 mL) were drawn into EDTA vacutainers both before medication dosing on days 0, 1, 6, and 7 (in the morning) and at 16 time points (hours 0.5, 1, 1.5, 2, 3, 4, 5, 6, 7, 8, 10, 12, 16, 24, 48, and 72) after final dosing on day 7. Immediately after sample collection, vacutainer tubes were centrifuged at 3000 rpm at 4° C. for 10 minutes; within 1 hour of collection, they were stored at −20° C. Plasma samples were analyzed for L-lysine-d-amphetamine and d-amphetamine using a validated LC/MS/MS method.

By dose 5, d-amphetamine reached steady state. After dose 7, mean AUC₀₋₂₄ was 1113 ng·h/mL, mean AUC_(0-∞) was 1453 ng·h/mL, mean C_(max) was 90.1 ng·h/mL, and mean T_(max) was 3.68 hours. See Table 63 and FIG. 60. In comparison, extended-release amphetamine salts exhibit a T_(max) of 5.8 hours and AUC_(0-∞) 853 ng·h/mL after an overnight fast. J. F. Auiler et al., “Effect of food on early drug exposure from extended-release stimulants: results from the Concerta, Adderall XR Food Evaluation (CAFE) study,” Curr Med Res Opin 18: 311-316 at 313 (2002).

Intact L-lysine-d-amphetamine was rapidly converted to d-amphetamine. After dose 7, mean AUC₀₋₂₄ was 60.66 ng·h/mL, and mean AUC_(0-∞) was 61.06 ng·h/mL. See Table 63 and FIG. 60. In addition, mean C_(max) was 47.9 ng·h/mL, and mean T_(max) was 1.14 hours for intact L-lysine-d-amphetamine. L-lysine-d-amphetamine was completely eliminated within approximately 6 hours.

The results also indicate that l-lysine-d-amphetamine administration leads to a decrease in patient to patient variability of amphetamine levels (see the lower CV values for the L-lysine-d-amphetamine conjugate as compared to d-amphetamine).

There were no gender differences in systemic exposure to d-amphetamine, though C_(max) was 12% higher in men after normalization by body weight.

The multidose pharmacokinetic profile of d-amphetamine released from the prodrug L-lysine-d-amphetamine is consistent with extended-release properties. The adverse events that occurred in this setting are consistent with other stimulants and suggest that suggest that L-lysine-d-amphetamine 70 mg is well tolerated.

TABLE 63 Steady-state pharmacokinetics parameters (n = 11) Parameter Mean SD CV % d-amphetamine C_(max) (ng/mL) 90.1 29.6 32.84 C_(min) (ng/mL) 18.2 14.2 78.12 T_(max) (h) 3.68 1.42 38.54 t_(1/2) (h) 10.08 2.76 27.37 AUC₀₋₂₄ (ng · h/mL) 1113 396.8 35.65 AUC_(0-∞) (ng · h/mL) 1453 645.7 44.45 AUC_(0-t) (ng · h/mL) 1371 633.5 46.19 FI (%) 163.55 37.20 22.74 Intact L-lysine-d-amphetamine C_(max) (ng/mL) 47.9 18.6 38.81 C_(min) (ng/mL) 0.0 0.0 — T_(max) (h) 1.14 0.32 28.45 t_(1/2) (h) 0.43 0.09 21.90 AUC₀₋₂₄ (ng · h/mL) 60.66 21.00 34.61 AUC_(0-∞) (ng · h/mL) 61.06 20.63 33.79 AUC_(0-t) (ng · h/mL) 59.44 21.47 36.12 FI (%) 1896.06 340.24 17.94

Example 33 Clinical Pharmacokinetic Evaluation and Oral Bioavailability of L-lysine-d-amphetamine Dimesylate Compared to Amphetamine Extended Release Products Adderall XR® and Dexedrine Spansule® Used in the Treatment of ADHD

TABLE 64 Treatment groups and dosage for clinical pharmacokinetic evaluation of L- lysine-d-amphetamine compared to Adderall XR ® or Dexedrine Spansule ® Treatment No. of Dose Dose Drug Group Subjects Dose (mg) (amphetamine base) L-lysine- A 10 1 × 25 mg 25 7.37 d-amphetamine capsule L-lysine- B 10 3 × 25 mg capsules 75 22.1 d-amphetamine Dexedrine C 10 3 × 10 mg capsules 30 22.1 Spansule ® Adderall XR ® D 10 1 × 30 mg capsules 35 21.9 plus 1 × 5 mg capsule

A clinical evaluation of the pharmacokinetics and oral bioavailability of L-lysine-d-amphetamine in humans was conducted. L-lysine-d-amphetamine was orally administered at doses approximating the lower (25 mg) and higher (75 mg) end of the therapeutic range based on d-amphetamine base content of the doses. Additionally, the higher dose was compared to doses of Adderall XR® (Shire) or Dexedrine Spansule® (GlaxoSmithKline) containing equivalent amphetamine base to that of the higher L-lysine-d-amphetamine dose. Treatment groups and doses are summarized in Table 64. All levels below limit quantifiable (blq <0.5 ng/mL) were treated as zero for purposes of pharmacokinetic analysis.

The concentrations of d-amphetamine and L-lysine-d-amphetamine intact conjugate following administration of L-lysine-d-amphetamine at the low and high dose for each individual subject as well as pharmacokinetic parameters are presented in Table 65-Table 70. The concentrations of d-amphetamine following administration of Adderall XR® or Dexedrine Spansule® for each individual subject as well as pharmacokinetic parameters are presented in Table 69 and Table 70, respectively. Concentration-time curves showing L-lysine-d-amphetamine intact conjugate and d-amphetamine are presented in FIG. 61 and FIG. 62. Extended release of d-amphetamine from L-lysine-d-amphetamine was observed for both doses and pharmacokinetic parameters (C_(max) and AUC) were proportional to doses when the lower and higher dose results were compared (FIG. 61 and FIG. 62). Significant levels of d-amphetamine were not observed until one-hour post administration. Only small amounts (1.6 and 2.0 percent of total drug absorption, respectively for 25 and 75 mg doses; AUC_(inf)-molar basis) of L-lysine-d-amphetamine intact conjugate were detected with levels peaking at about one hour (Table 66 and Table 68). The small amount of intact conjugate absorbed was rapidly and completely eliminated, with no detectable concentrations present by five hours, even at the highest dose.

In a cross-over design (identical subjects received Adderall XR® doses following a 7-day washout period), the higher L-lysine-d-amphetamine dose was compared to an equivalent dose of Adderall XR®. Adderall XR® is a once-daily extended release treatment for ADHD that contains a mixture of d-amphetamine and l-amphetamine salts (equal amounts of d-amphetamine sulfate, d-/l-amphetamine sulfate, d-amphetamine saccharate, and d-/l-amphetamine aspartate). An equivalent dose of extended release Dexedrine Spansule® (contains extended release formulation of d-amphetamine sulfate) was also included in the study. As observed in pharmacokinetic studies in rats, oral administration of L-lysine-d-amphetamine resulted in d-amphetamine concentration-time curves similar to those of Adderall XR® and Dexedrine Spansule® (FIG. 63 and FIG. 64). The bioavailability (AUC_(inf)) of d-amphetamine following administration of L-lysine-d-amphetamine was approximately equivalent to both extended release amphetamine products (Table 71). Over the course of twelve hours, typically the time needed for effective once-daily treatment of ADHD, the bioavailability for L-lysine-d-amphetamine was approximately equivalent to that of Adderall XR® (d-amphetamine plus l-amphetamine levels) and over twenty percent higher than that of Dexedrine Spansule®. Based on the results of this clinical study, L-lysine-d-amphetamine would be an effective once-daily treatment for ADHD. Moreover, L-lysine-d-amphetamine afforded similar pharmacokinetics in humans and animal models, namely, delayed release of d-amphetamine resulting in extended release kinetics. Based on these observations L-lysine-d-amphetamine should also have abuse-resistant properties in humans.

TABLE 65 Individual subject d-amphetamine concentrations and pharmacokinetic parameters following oral administration of a 25 mg dose of L-lysine-d-amphetamine to humans Subject Subject Subject Subject Subject Subject Subject Subject Subject Subject 102 103 105 107 110 112 113 116 117 120 Mean SD CV % Time Hours  0 0 0 0 0 0 0 0 0 0 0 0 0 0  0.5 0 0 0.625 0 0 0 0 0.78 0.769 0 0.2 0.4 162.1  1 4.29 2.95 8.67 3.36 8.33 1.1 10 10.5 14 3.15 6.6 4.2 63.6  1.5 10 12.7 16 13.8 21.4 3.94 24.7 19.5 24 15.1 16.1 6.5 40.3  2 16.3 18.4 17 21 25.9 9.29 30.9 23.6 30 21.7 21.4 6.6 30.8  3 16.5 19.6 16.7 26.1 27 17.7 30.2 23.5 27.6 28.9 23.4 5.3 22.7  4 23.9 18.8 14.1 24.5 30.1 17.9 33.2 21.2 24.7 25.3 23.4 5.7 24.3  5 21.2 18.9 14.6 21.6 22.6 17.2 27 20 20.2 24.2 20.8 3.5 16.9  6 21.8 18 12.5 21.6 23.7 15.7 25.8 18.2 20.3 20.5 19.8 3.9 19.6  7 18.9 15.8 12.1 17.8 20.6 14.5 26.6 21 18.3 21.8 18.7 4.1 21.9  8 19.3 16.6 10.4 17.9 20 14.2 25.7 13.6 18.8 20.1 17.7 4.2 24.1 10 18.8 13.6 9.8 15.3 19.3 13.7 22.4 15.1 15.3 15.9 15.9 3.5 22.1 12 15.8 12.6 6.92 11.5 15.8 11.2 17.9 12 13.7 15.2 13.3 3.1 23.6 16 13.4 10.5 6.56 9.53 14.3 10.7 12.5 10.3 10 13 11.1 2.3 20.5 24 8.03 5.81 2.65 4.9 5.8 5.9 6.57 6.13 4.52 5.45 5.6 1.4 25.1 48 1.57 1.36 0 1.26 0.795 1.44 1.24 1.23 0.864 0.586 1.0 0.5 46.1 72 0 0 0 0 0 0 0 0 0 0 0 0 0 Parameter AUC_(0-12 h) 204.0 177.4 140.4 204.9 242.7 152.4 284.6 199.2 225.5 223.3 205.4 42.5 20.7 (ng · h/mL) AUC_(last) 463.3 375.1 201.4 378.5 462.7 350.7 515.2 397.9 395.7 426.1 396.7 84.8 21.4 (ng · h/mL) AUC_(inf) 486.7 397.1 233.5 398.8 472 374 532.5 416.4 407 432.2 415.0 80.1 19.3 (ng · h/mL) C_(max) 23.9 19.6 17 26.1 30.1 17.9 33.2 23.6 30 28.9 25.0 5.6 22.3 (ng/mL) T_(max) (hours) 4 3 2 3 4 4 4 2 2 3 3.1 0.876 28.2 T_(1/2) (hours) 10.32 11.18 8.36 11.18 8.16 11.22 9.68 10.43 9.06 7.22 9.68 1.43 14.7

TABLE 66 Individual subject L-lysine-d-amphetamine intact conjugate concentrations and pharmacokinetic parameters following oral administration of a 25 mg dose of L-lysine-d-amphetamine to humans Subject Subject Subject Subject Subject Subject Subject Subject Subject Subject 102 103 105 107 110 112 113 116 117 120 Mean SD CV % Time Hours  0 0 0 0 0 0 0 0 0 0 0 0 0 0  0.5 4.1 5.5 10.0 0.0 3.6 0.0 9.2 9.6 8.9 0.0 5.1 4.2 82.0  1 9.2 11.2 15.2 12.5 9.1 2.7 20.1 10.5 10.8 10.9 11.2 4.5 39.7  1.5 4.0 4.4 6.1 7.5 3.6 6.2 6.6 2.8 4.2 8.4 5.4 1.8 34.1  2 2.1 1.4 2.5 2.9 1.9 4.0 2.3 0 1.7 3.1 2.2 1.1 48.8  3 0 0 0 0 0 0 0 0 0 0 0 0 0  4 0 0 0 0 0 0 0 0 0 0 0 0 0  5 0 0 0 0 0 0 0 0 0 0 0 0 0  6 0 0 0 0 0 0 0 0 0 0 0 0 0  7 0 0 0 0 0 0 0 0 0 0 0 0 0  8 0 0 0 0 0 0 0 0 0 0 0 0 0 10 0 0 0 0 0 0 0 0 0 0 0 0 0 12 0 0 0 0 0 0 0 0 0 0 0 0 0 16 0 0 0 0 0 0 0 0 0 0 0 0 0 24 0 0 0 0 0 0 0 0 0 0 0 0 0 48 0 0 0 0 0 0 0 0 0 0 0 0 0 72 0 0 0 0 0 0 0 0 0 0 0 0 0 Parameter AUC_(last) 9.18 10.95 16.31 10.68 8.583 5.439 18.51 10.77 12.35 10.41 11.32 3.74 33.1 (ng · h/mL) AUC_(inf) 10.62 11.64 17.66 12.65 9.759 — 19.56 — 13.3 12.83 13.50 3.40 25.2 (ng · h/mL) C_(max) (ng/mL) 9.18 11.2 15.2 12.5 9.05 6.18 20.1 10.5 10.8 10.9 11.56 3.80 32.9 T_(max) (hours) 1 1 1 1 1 1.5 1 1 1 1 1.05 0.16 15.1 T_(1/2) (hours) 0.47 0.34 0.38 0.47 0.44 — 0.32 — 0.38 0.55 0.419 0.077 18.5

TABLE 67 Individual subject d-amphetamine concentrations and pharmacokinetic parameters following oral administration of a 75 mg dose of L-lysine-d-amphetamine to humans Subject Subject Subject Subject Subject Subject Subject Subject Subject Subject 101 104 106 108 109 111 114 115 118 119 Mean SD CV % Time Hours  0 0 0 0 0 0 0 0 0 0 0 0 0 0  0.5 0 0.748 0.506 0 0 0.779 0.525 0 3 1.85 0.7 1.0 132.2  1 11.9 14.4 12.6 7.26 5.9 10.3 7.2 23.1 23 27.9 14.4 7.7 53.6  1.5 40.3 34.6 30.4 22.8 19.3 38.4 19 52.8 51.5 55.8 36.5 13.8 37.8  2 84.6 48.9 68.2 34.8 32.7 57.2 33.1 91.3 61.7 70.4 58.3 21.0 36.0  3 72.9 64.3 55.7 60.3 62.3 61.1 44.8 95.8 62.1 83.6 66.3 14.5 21.9  4 84.6 65.3 58.8 51.1 77.9 63.3 47.6 89.2 54.2 86 67.8 15.5 22.8  5 65 55.6 60.2 74 83.9 59.1 56.9 77.7 54.9 82.8 67.0 11.5 17.2  6 71 53.5 49.4 51.5 78.3 50.8 55.1 68.8 52.9 64 59.5 10.2 17.1  7 53.8 55.7 52.9 69.5 73.1 52.9 55.9 71.2 45.1 74.6 60.5 10.5 17.4  8 63.7 40.3 47.3 45.7 72.2 46.5 54.2 61.1 44.3 66.2 54.2 10.9 20.2 10 43.7 41.7 37 58.4 67 44.3 48.4 68 34.1 55.9 49.9 11.9 24.0 12 46.4 26.1 36.7 37.4 49.9 32.4 37.1 54.1 34.5 45.1 40.0 8.6 21.6 16 35.4 22.2 25.7 48 44.9 24.3 28.9 44.7 31.7 34.5 34.0 9.2 27.1 24 16.4 11.4 14.9 13.2 18.4 16.8 20.5 21.7 15.7 18.1 16.7 3.1 18.8 48 2.74 2.14 4.17 2.73 3.75 4.81 2.81 4.26 3.36 3.4 0.9 25.9 72 0 0 0 1.07 0.661 0.687 1.49 0 0 0.553 0.4 0.5 120.2 Parameter AUC_(0-12 h) 666.2 525.9 531.6 570.3 704.8 545.6 513.7 790.9 523.4 742.8 611.5 104.5 17.1 (ng · h/mL) AUC_(last) 1266 918.7 1031 1257 1442 1123 1223 1549 1143 1417 1237.0 194.0 15.7 (ng · h/mL) AUC_(inf) 1301 948.3 1072 1278 1451 1133 1251 1582 1154 1425 1259.5 191.3 15.2 (ng · h/mL) C_(max) (ng/mL) 84.6 65.3 68.2 74 83.9 63.3 56.9 95.8 62.1 86 74.0 12.9 17.4 T_(max) (hours) 4 4 2 5 5 4 5 3 3 4 3.9 1.0 25.5 T_(1/2) (hours) 8.78 9.59 10.02 13.26 9.24 10.41 12.8 8.05 10.92 9.47 10.3 1.7 16.3

TABLE 68 Individual subject L-lysine-d-amphetamine intact conjugate concentrations and pharmacokinetic parameters following oral administration of a 75 mg dose of L-lysine-d-amphetamine to humans Subject Subject Subject Subject Subject Subject Subject Subject Subject Subject 101 104 106 108 109 111 114 115 118 119 Mean SD CV % Time Hours  0 0 0 0 0 0 0 0 0 0 0 0 0 0  0.5 10.4 22.6 6.92 10.3 0 9.21 7.88 14.5 87.8 35.5 20.5 25.6 124.7  1 48 40.5 29 41.5 21.2 30.8 23.4 127 88.9 80.1 53.0 34.6 65.2  1.5 28.4 15.7 16.1 20.3 26.5 19 12.7 38.7 28.6 38 24.4 9.2 37.5  2 8.87 5.53 4.91 9 18.1 5.62 6.29 12.1 9.75 11.3 9.1 4.0 44.0  3 2.15 1.29 1.76 1.82 10.6 0 2.31 2.57 1.73 1.73 2.6 2.9 111.6  4 0 0 1.09 0 4.65 0 1.53 1.01 0 0 0.8 1.5 176.9  5 0 0 0 0 0 0 0 0 0 0 0 0 0  6 0 0 0 0 0 0 0 0 0 0 0 0 0  7 0 0 0 0 0 0 0 0 0 0 0 0 0  8 0 0 0 0 0 0 0 0 0 0 0 0 0 10 0 0 0 0 0 0 0 0 0 0 0 0 0 12 0 0 0 0 0 0 0 0 0 0 0 0 0 16 0 0 0 0 0 0 0 0 0 0 0 0 0 24 0 0 0 0 0 0 0 0 0 0 0 0 0 48 0 0 0 0 0 0 0 0 0 0 0 0 0 72 0 0 0 0 0 0 0 0 0 0 0 0 0 Parameter AUC_(last) 51.2 44.2 32.0 43.7 50.4 30.9 29.8 102.1 110.8 86.1 58.1 30.2 52.0 (ng · h/mL) AUC_(inf) 52.5 45.0 33.0 44.9 52.3 34.2 31.4 102.9 111.7 87.0 59.5 29.9 50.2 (ng · h/mL) C_(max) 48.0 40.5 29.0 41.5 26.5 30.8 23.4 127.0 88.9 80.1 53.6 34.1 63.6 (ng/mL) T_(max) (hours) 1 1 1 1 1.5 1 1 1 1 1 1.05 0.16 15.1 T_(1/2) (hours) 0.43 0.4 0.61 0.43 1.02 0.41 0.75 0.56 0.38 0.35 0.534 0.211 39.6

TABLE 69 Individual subject d-amphetamine concentrations and pharmacokinetic parameters following oral administration of a 35 mg dose of Adderall XR ® (equivalent to 75 mg dose of L-lysine-d-amphetamine based on amphetamine base content) to humans Subject Subject Subject Subject Subject Subject Subject Subject Subject Subject 101 104 106 108 109 111 114 115 118 119 Mean SD CV % Time Hours  0 0 0 0 0 0 0 0 0 0 0 0 0 0  0.5 7.9 2.3 2.8 0.6 2.2 5.7 0 16 2.3 5.3 4.5 4.7 104.3  1 37.6 28.9 23.3 13.7 29.8 38.2 17.9 46.2 28.8 48.8 31.3 11.5 36.6  1.5 49.9 42.3 31.1 23.7 39.1 34.4 30.8 65.4 34.1 53 40.4 12.5 31.0  2 65.9 45.8 29.2 37.4 46.2 65.4 40 64.4 37 67.8 49.9 14.6 29.2  3 95.3 51.7 36.7 23.6 64.7 62.9 44.7 56.5 31.1 64.8 53.2 20.7 38.9  4 83.7 73.3 56.7 40 67 76.6 56.3 53.1 33.5 73.3 61.4 16.3 26.6  5 77.4 75.2 71.6 62.1 75.9 76.4 51.5 61.4 56.8 82.4 69.1 10.3 14.9  6 71.5 72.1 64 59.8 66.9 63.5 56.8 59.8 58.7 85.7 65.9 8.7 13.2  7 72.3 63.6 71 57.9 70.6 69.7 51.9 48.1 53.7 79.7 63.9 10.5 16.4  8 60.4 57.1 53.8 53 72 66.9 56.2 56.4 51.7 66.7 59.4 6.9 11.6 10 50.4 45.5 53 50.7 67.6 57.4 49.1 66.6 48 71.3 56.0 9.3 16.6 12 42.5 41.3 45.4 32.9 53.1 46 37.3 74.7 42.2 60.2 47.6 12.2 25.7 16 31.1 29.6 35.7 39 45.2 33.9 34.3 64.9 29 40.5 38.3 10.6 27.7 24 14.9 15.1 22.1 19.5 21.7 21.2 20.7 35.7 17.9 20.5 20.9 5.8 27.7 48 2.5 4.2 3.8 5.9 5.4 3.8 7.3 5.1 3.9 3 4.5 1.4 32.1 72 0 0.3 1 1 0.3 1.1 2.7 0.3 0 0 0.7 0.8 124.7 Parameter AUC_(0-12 h) 731.2 625.0 582.6 504.3 711.6 698.5 535.4 683.5 509.8 793.2 637.5 101.1 15.9 (ng · h/mL) AUC_(last) 1270 1230 1343 1269 1568 1436 1354 1920 1101 1520 1401.1 229.0 16.3 (ng · h/mL) AUC_(inf) 1301 1234 1358 1286 1571 1454 1418 1923 1164 1557 1426.6 218.9 15.3 (ng · h/mL) C_(max) 95.3 75.2 71.5 62 75.9 76.5 56.8 74.7 58.8 85.8 73.3 11.9 16.3 (ng/mL) T_(max) (hours) 3 5 5 5 5 4 6 12 6 6 5.70 2.41 42.2 T_(1/2) (hours) 8.65 9.01 10.57 11.58 8.37 10.78 16.4 7.25 11.05 8.54 10.22 2.59 25.3

TABLE 70 Individual subject d-amphetamine concentrations and pharmacokinetic parameters following oral administration of a 30 mg dose of Dexedrine Spansule ® (equivalent to 75 mg dose of L-lysine-d-amphetamine based on amphetamine base content) to humans Subject Subject Subject Subject Subject Subject Subject Subject Subject Subject 102 103 105 107 110 112 113 116 117 120 Mean SD CV % Time Hours  0 0 0 0 0 0 0 0 0 0 0 0 0 0  0.5 1.2 2.68 1.37 1.4 1.16 2.36 6.75 2.63 4.95 3.43 2.8 1.8 65.5  1 14.8 26.5 16.7 21.4 25.2 12.7 33.1 22.3 26 21.5 22.0 6.1 27.8  1.5 24.2 36.9 23.2 28.5 37.2 21.3 42.4 29.2 33.7 39.2 31.6 7.3 23.2  2 28.6 43.4 27.3 34.6 38.5 27.6 46.2 31.3 38.5 42 35.8 6.9 19.4  3 27.4 37.3 30.6 40.1 41.7 30.9 52 36.5 42.9 60.1 40.0 10.0 25.2  4 27.1 44.1 33.5 48.7 45.2 34.7 49.1 40.7 42.4 53.2 41.9 8.1 19.2  5 35.1 53 40.2 43.4 46.5 42.4 58.1 47 52.1 68.7 48.7 9.7 20.0  6 33.8 58.5 40.2 46.5 43.5 37.5 56.2 40 51 63 47.0 9.8 20.8  7 37.2 50.7 31.2 41.4 44.9 42 57.8 43.6 51.6 65.7 46.6 10.1 21.7  8 35.9 54.3 34.9 45 45 36 58.7 41.8 53.9 59.2 46.5 9.5 20.4 10 33.1 49.1 34.3 35.5 45 37 51.4 38.9 46.3 60.1 43.1 8.8 20.4 12 34 51 28.6 34.1 40.8 32.6 51.6 37.7 38.1 50.9 39.9 8.4 21.1 16 30.2 40.8 25.2 28 33 25.8 41 26.8 29.6 44.9 32.5 7.1 22.0 24 20.5 27.8 18.2 19.5 17.1 17.8 22.5 19.1 15.5 27.3 20.5 4.2 20.3 48 3.83 6.89 3.7 5.11 2.56 4.31 6.51 4.43 2.77 5.47 4.6 1.4 31.8 72 0.715 1.63 1 1.7 0 0.622 1.29 1.22 0 1.31 0.9 0.6 64.0 Parameter AUC_(0-12 h) 356.2 539.8 366.4 444.3 480.8 387.0 591.4 436.5 512.8 634.2 474.9 94.7 19.9 (ng · h/mL) AUC_(last) (ng · h/mL) 1033 1517 966 1135 1065 1003 1473 1100 1048 1589 1193 236 19.8 AUC_(inf) (ng · h/mL) 1043 1544 983.5 1168 1097 1013 1495 1121 1085 1610 1216 238 19.5 C_(max) (ng/mL) 37.2 58.5 40.2 48.7 46.5 42.4 58.7 47 53.9 68.7 50.18 9.74 19.4 T_(max) (hours) 7 6 5 4 5 5 8 5 8 5 5.80 1.40 24.1 T_(1/2) (hours) 9.92 11.74 12.07 13.8 8.7 10.76 11.47 12.23 9.36 10.92 11.10 1.50 13.6

TABLE 71 Pharmacokinetic parameters of amphetamine following oral administration of L-lysine-d-amphetamine, Adderall XR ® or Dexedrine Spansule ®. Drug L-lysine- L-lysine- d- d- amphetamine amphetamine Adderall Dexedrine Parameter 25 mg Percent¹ 75 mg Percent¹ XR ® Percent¹ Spansule ® Percent¹ AUC_(0-12 h) 205.4 33.6 611.5 100  637.5 104 474.9 78 (ng · h/mL) AUC_(last) (ng · h/mL) 396.7 31.5 1237 100 1401.1 113 1193 96 AUC_(inf) (ng · h/mL)  415 ± 80.3 32.9 1260 ± 192  100 1429 ± 223  113 1217 ± 237  97 C_(max) (ng/mL) 25.0 ± 5.57 33.8 74.0 ± 12.9 100 73.3 ± 11.9 99 50.2 ± 9.74 68 T_(max) (hours) 3.10 ± 0.88 79.5 3.90 ± 0.99 100 5.70 ± 2.41 146  5.8 ± 1.40 149 T_(1/2) (hours) 9.66 ± 1.45 94 10.3 ± 1.66 100 10.2 ± 2.62 99 11.1 ± 1.48 108 ¹Percent relative to L-lysine-d-amphetamine 75 mg dose

Example 34 Clinical Pharmacokinetic Evaluation and Oral Bioavailability of L-lysine-d-amphetamine Dimesylate

In pediatric patients (6-12 yrs) with ADHD, the T_(max) of d-amphetamine was approximately 3.5 hours following single-dose oral administration of L-lysine-d-amphetamine dimesylate either 30 mg, 50 mg, or 70 mg after a 8-hour overnight fast. See FIG. 65. The T_(max) of L-lysine-d-amphetamine dimesylate was approximately 1 hour. Linear pharmacokinetics of d-amphetamine after single-dose oral administration of L-lysine-d-amphetamine dimesylate was established over the dose range of 30 mg to 70 mg in children.

TABLE 72 Pharmacokinetic parameters of d-amphetamine and L-lysine-d-amphetamine dimesylate C_(max) T_(max) AUC t_(1/2) Dose (ng/mL) (h) (ng · h/mL) (h) d-amphetamine 30 mg 53.2 ± 9.62 3.41 ± 1.09  845 ± 117 8.90 ± 1.33 50 mg 93.3 ± 18.2 3.58 ± 1.18 1510 ± 242 8.61 ± 1.04 70 mg  134 ± 26.1 3.46 ± 1.34 2157 ± 383 8.64 ± 1.32 Intact L-lysine-d-amphetamine dimesylate 30 mg 21.9 0.97 27.9 50 mg 46.0 0.98 57.9 70 mg 89.5 1.07 108.9

There is no unexpected accumulation of d-amphetamine at steady state in children with ADHD and no accumulation of L-lysine-d-amphetamine dimesylate after once-daily dosing for 7 consecutive days.

Food does not affect the extent of absorption of d-amphetamine in healthy adults after single-dose oral administration of 70 mg of L-lysine-d-amphetamine dimesylate capsules but delays T_(max) by approximately 1 hour (from 3.78 hrs at fasted state to 4.72 hrs after a high fat meal). After an 8-hour fast, the extent of absorption of d-amphetamine following oral administration of L-lysine-d-amphetamine dimesylate in solution and as intact capsules was equivalent.

There were no apparent differences between males and females in exposure as measured by dose-normalized C_(max) and AUC although the range of values in children was higher than that in adults. This is a consequence of the significant correlation between dose-normalized C_(max) and AUC and body weight and thus the differences are due to the higher doses in mg/kg administered to children. There were no apparent differences in t_(1/2) between male and female subjects nor were there any apparent relationships between t_(1/2) and either age or body weight.

Exemplary results of clinical pharmacokinetic evaluation are presented in FIG. 66 (AUC), FIG. 67 (C_(max)), and FIG. 68 (T_(max)).

Example 35 Efficacy of L-lysine-d-amphetamine Dimesylate in Pediatric Clinical Trials

The efficacy of L-lysine-d-amphetamine dimesylate was established in a double-blind, randomized, placebo-controlled, parallel-group study conducted in children aged 6-12 (N=290) who met DSM-IV criteria for ADHD (either the combined type or the hyperactive-impulsive type). Patients were randomized to fixed dose treatment groups receiving final doses of 30, 50, or 70 mg of L-lysine-d-amphetamine dimesylate or placebo once daily in the morning for four weeks. For patients randomized to 50 and 70 mg L-lysine-d-amphetamine dimesylate, dosage was increased by forced titration. Significant improvements in the signs and symptoms of ADHD, as rated by investigators (ADHD Rating Scale; ADHD-RS) and parents (Connor's Parent Rating Scale; CPRS), were demonstrated for all L-lysine-d-amphetamine dimesylate doses compared to placebo, for all four weeks, including the first week of treatment, when all L-lysine-d-amphetamine dimesylate patients were receiving a dose of 30 mg/day. Additional dose-responsive improvement was demonstrated in the 50 and 70 mg groups, respectively. L-lysine-d-amphetamine dimesylate-treated patients showed significant improvements, as measured by CPRS scores, in the morning (˜10 am), afternoon (˜2 pm), and evening (˜6 pm) compared with placebo-treated patients, demonstrating effectiveness throughout the day. The results of the primary efficacy analysis, ADHD-RS total score change from baseline to endpoint for the ITT population, are shown in FIG. 69.

Efficacy was also measured by the SKAMP score. A total of 52 children ages 6 to 12 who met DSM-IV criteria for ADHD (either the combined type or the hyperactive-impulsive type) were enrolled in a double-blind, randomized, placebo-controlled crossover study. Patients were randomized to receive fixed and optimal doses of L-lysine-d-amphetamine (30, 50, 70 mg), Adderall XR® (10, 20, or 30 mg), or placebo once daily in the morning for 1 week each treatment. The primary efficacy endpoint in this study was SKAMP-Deportment score (Swanson, Kotkin, Agler, M. Flynn and Pelham rating scale). Both L-lysine-d-amphetamine and Adderall XR® were highly effective compared to placebo. The significant effects of L-lysine-d-amphetamine occurred within 2 hours post morning dose and continued throughout the last assessment time point, 12 hours post morning dose, compared to placebo, yielding a 12-hour duration of action. See FIG. 70.

Example 36 Abuse Liability of Intravenous L-lysine-d-amphetamine

L-lysine-d-amphetamine 50 mg, d-amphetamine 20 mg, and placebo were given intravenously over 2 minutes at 48 hour intervals to 9 stimulant abusers in a double blind crossover design to assess abuse liability. Drugs were given according to 3×3 balanced latin squares. Each dosing day, vital sign measures and subjective and behavioral effects were assessed with questionnaires before dosing and at 0.5, 1, 1.5, 2, 3, 4, 5, 6, 9, 12, 16 and 24 hours after dosing. At these times and at 5 minutes, a blood sample (5 ml) was taken for d-amphetamine levels.

For d-amphetamine, mean peak plasma level of 77.7 ng/ml of d-amphetamine occurred at 5 minutes and then rapidly subsided. Administration of d-amphetamine produced expected d-amphetamine-like effects with mean peak responses at 15 minutes. The mean maximum response to d-amphetamine on the primary variable of Subject Liking VAS was significantly greater than placebo (p=0.01).

For L-lysine-d-amphetamine, mean peak plasma level of 33.8 ng/ml of d-amphetamine occurred at 3 hours and remained at this level through the 4 hour observation. L-lysine-d-amphetamine produced d-amphetamine-like subjective, behavioral, and vital sign effects with mean peak responses at 1 to 3 hours. For the primary variable of Subject Liking VAS, the response was not greater than placebo (p=0.29). Changes in blood pressure following L-lysine-d-amphetamine were significant.

At the end of the study, subjects were asked which treatment they would take again. Six subjects chose d-amphetamine 20 mg, two subjects chose none of the treatments, and one subject chose L-lysine-d-amphetamine 50 mg. In summary, L-lysine-d-amphetamine 50 mg did not produce euphoria or amphetamine-like subjective effects although there were late occurring blood pressure increases. The findings suggest that L-lysine-d-amphetamine itself is inactive. After 1 to 2 hours, L-lysine-d-amphetamine is converted to d-amphetamine. Taken intravenously, L-lysine-d-amphetamine has significantly less abuse potential than immediate release d-amphetamine containing an equal amount of d-amphetamine base.

Example 37 Preliminary Estimates of Decreased Abuse Liability with L-lysine-d-amphetamine Vs. d-amphetamine in Healthy Adults with a History of Stimulant Abuse

This randomized, single-center, single-blind, dose-escalation study used pharmacokinetic parameters to obtain preliminary estimates of abuse liability for L-lysine-d-amphetamine (30-150 mg) vs. d-amphetamine sulfate (40 mg) and placebo in healthy adults meeting DSM-IV criteria for stimulant abuse. Subjects were divided into 3 cohorts of 4 patients each; all received single doses of L-lysine-d-amphetamine at a minimum interval of 48 hours, with d-amphetamine sulfate (40 mg) and placebo randomly dispersed. Cohort 1 was administered L-lysine-d-amphetamine doses of 30, 50, 70, 100 mg; cohort 2 received 50, 70, 100, 130 mg doses; and cohort 3 received 70, 100, 130, and 150 mg doses.

AUC_(last) d-amphetamine over the first 4 hours was substantially lower with 100 mg L-lysine-d-amphetamine (165.3-213.1 ng/mL) vs. 40 mg d-amphetamine (245.5-316.8 ng/mL). C_(max) and AUC_(last) increased with dose for 30-130 mg L-lysine-d-amphetamine, attenuating between the 130 mg and 150 mg dose. T_(max) ranged from 3.78-4.25 h with L-lysine-d-amphetamine vs. d-amphetamine sulfate (1.88-2.74 h). The half-life of L-lysine-d-amphetamine (range, 0.44-0.76 h) indicated rapid clearance. Adverse reactions were mild in severity with no significant changes in vital signs or ECG parameters. L-lysine-d-amphetamine had a slower release of d-amphetamine compared with d-amphetamine sulfate. At doses as high as 150 mg, there appears to be an attenuation of the maximum concentration, suggesting higher doses of L-lysine-d-amphetamine will not lead to further increases in C_(max) and AUC_(last). These results suggest a drug profile consistent with reduced abuse liability.

Example 38 Preparation of L-lysine-d-amphetamine Dimesylate

L-lysine-d-amphetamine dimesylate was prepared according to the procedure shown in FIG. 2 and as described in more detail below.

L-Lysine monohydrochloride (1 wt=1 eq) and water (6 vol) were added to a clean vessel and then heated to ˜35° C. The pH of the mixture was adjusted to ˜11-11.5 using 50% sodium hydroxide solution. Di-tert-butyl dicarbonate and 50% sodium hydroxide solution were charged to the vessel at a rate such that the temperature was maintained at ˜30-50° C. and pH>8.

The reaction mixture was then heated to ˜50° C. and the temperature was maintained for 2 hr., or until reaction completion. The mixture was cooled to ˜20-25° C., followed by dilution with isopropyl acetate (IPAc) (6 vol.). The pH of the mixture was adjusted to ˜2.0-2.5 using concentrated hydrochloric acid. Then, the mixture was agitated for 15 min. followed by phase separation. The organic phase was washed with brine (25% wt/wt, 1.34 vol.).

The mixture containing the non-isolated intermediate Boc-L-Lys(Boc)-OH was charged on top of a N-hydroxysuccinimide solution (1.1 eq.) in a clean vessel. The original vessel was then rinsed with IPAc (2.5 vol.) and the washings were combined with the mixture. The temperature of the mixture was adjusted to ˜50° C. A solution of N,N′-Dicyclohexyl carbodiimide (1.05 eq.) and IPAc (1.2 vol.) heated to ˜30° C. was then added. The temperature of the reaction mixture was maintained at ˜50° C. The mixture was then agitated for 2 hr., or until reaction completion. The mixture was cooled to ˜20-25° C. followed by filtration to remove N,N′-dicyclohexylurea.

Following filtration, the filter-cake was rinsed with IPAc (4 vol.). The filtrate (IPAc solution) was washed twice with saturated sodium bicarbonate solution (1.3 vol. per wash). The IPAc solution was washed with water (1.2 vol.) and then brine (25% wt/wt, 1.34 vol.). The reaction mixture was concentrated to 12 vol. by vacuum distillation below 50° C. to give the non-isolated intermediate Boc-L-Lys(Boc)-OSu.

d-amphetamine (1.1 eq.), 4-methylmorpholine (1.1 eq) and IPAc (12 vol.) were charged into a clean vessel. The solution of Boc-L-Lys(Boc)-OSu in IPAc was then charged into the same vessel at a rate such that the temperature remained below 30° C. The reaction mixture was agitated for 2 hr. at ˜20-30° C., or until reaction completion.

A quench solution was prepared by mixing sodium chloride (4.5 wt), glacial acetic acid (0.3 wt) and water (18 vol), and agitating it until dissolution was achieved. One half of the quench solution was added to the reaction mixture. The reaction mixture was agitated for 30 min., followed by phase separation. The remaining quench solution was added. The reaction mixture was washed with aqueous sodium bicarbonate solution (8% wt/wt, 11.5 vol.), followed by brine (7% wt/wt, 11.5 vol.). ˜6.25 vol. was removed from the reaction mixture by vacuum distillation at ˜55-65° C. Isopropyl alcohol (IPA) (21 vol.) was added. The mixture was concentrated to 6.25 vol. by vacuum distillation at ˜55-65° C. IPA (2.5 vol.) was again added, and the reaction mixture was filtered through a 1.2 mm filter. Methane sulfonic acid (2.05 eq.) and water (0.6 vol.) were added to the filtrate and the mixture was heated to ˜60-70° C. for 6 hr. at a partial vacuum (˜550 mmHg). When complete, pre-heated (60-70° C.) IPAc (7.7 vol.) was added to the mixture while the temperature was maintained at 60-70° C. The mixture was then agitated at 60-70° C. for 1-2 hrs., followed by cooling to 20-25° C. by lowering the jacket temperature ˜10° C./hour. The temperature was adjusted to 50-60° C. for 2-3 hours. The mixture was then cooled to 20-25° C. by lowering the jacket temperature ˜10° C./hour and aged at 20-25° C. for at least 16 hr.

The product was collected by filtration. The filter-cake was washed twice with a mixture of IPA and IPAc (2:1, 10.5 vol per wash), and dried at ˜50-60° C. in vacuo. The dried material was screened through a screener/magnet (20-mesh) to yield the final crystalline product.

Optical micrographs and SEM (scanning electron microscopy) images of the crystals prepared are shown in FIGS. 71 and 72, respectively.

Particle size determination provided d₁₀, d₅₀ and d₉₀ values of 5 μm, 26 μm, and 57 μm, respectively.

Example 39 Preparation of L-lysine-d-amphetamine Dimesylate

L-lysine-d-amphetamine dimesylate was prepared according to the procedure shown in FIG. 87 and as described in more detail below.

A. Preparation of d-amphetamine Free Base

D-amphetamine sulfate USP (1 eq.) and water (˜138 eq.) were added to a vessel and agitated to dissolve the solids. The pH of the mixture was adjusted to 10.5-11.5 using 50% sodium hydroxide solution. The mixture was diluted with IPAc (˜13.3 eq.). The mixture was agitated for 30-45 min., followed by phase separation. IPAc (˜2.7 eq.) was then added to the aqueous phase. The mixture was agitated for 30-45 min. and the phases were then again separated. The organic phase was combined with the organic phase from the previous phase separation. If the water content of the combined dehydrated organic phase was >4.0%, water (˜17.3 eq.) and sodium chloride (˜1.8 eq.) were added to the organic phase; the mixture was agitated for 30-45 min., the phases separated, and the dehydrated organic phase was then used for further processing.

B. Preparation of Boc-L-Lys(Boc)-OSu

L-Lysine monohydrochloride (1 wt.=1 eq.) and water (6 vol.) were heated to ˜35° C. in a clean vessel. The pH of the mixture was adjusted to approximately 11-11.5 using 50% sodium hydroxide solution. Di-tert-butyl dicarbonate and 50% sodium hydroxide solution were then charged to the vessel at a rate allowing to maintain the mixture temperature at approximately 30-60° C. and the pH between 8.5-10. The reaction mixture was heated to approximately 50° C. and the temperature was maintained for 2 hr., or until reaction completion. The mixture was subsequently cooled to approximately 20-25° C., and diluted with IPAc (6 vol.). The pH was then adjusted to approximately 2.0-2.5 using concentrated hydrochloric acid. The mixture was agitated for 30-45 min. and phases were then separated. The organic phase was washed with brine (25% wt/wt, 1.34 vol.). N-hydroxysuccinimide (1.1 eq.) was charged into a clean vessel. The mixture containing the non-isolated intermediate Boc-L-Lys(Boc)-OH was charged on top of the N-hydroxysuccinimide. The original vessel was rinsed with IPAc (2.5 vol.) and the washings were combined with the mixture. After which, the mixture temperature was adjusted to approximately 50° C.

N,N′-Dicyclohexyl carbodiimide (1.05 eq.) was charged to the mixture while maintaining the temperature at approximately 50° C. IPAc (3.7 vol.) was charged on top of the mixture at a rate allowing to maintain the mixture temperature at approximately 50° C. The mixture was agitated for at least 2 hr., or until reaction completion. The mixture was cooled to approximately 20-25° C. The mixture was filtered to remove N,N′-dicyclohexylurea. The filter-cake was rinsed with IPAc (5.9 vol.). The filtrate (IPAc solution) was washed with saturated sodium bicarbonate solution (2.6 vol.). The IPAc solution was washed with water (1.2 vol.) and then brine (25% wt/wt, 1.34 vol.).

The mixture was then concentrated to 25-65% of the starting volume via vacuum distillation below 35° C. to give the non-isolated intermediate Boc-L-Lys(Boc)-Osu. The mixture was filtered through a filter dressed with diatomaceous earth impregnated pads.

C. Preparation of L-lysine-d-amphetamine Dimesylate

Freebased d-amphetamine (1.1 eq.), 4-methylmorpholine (1.085 eq.) and IPAc (12 vol.) were added to a clean vessel. The solution of Boc-L-Lys(Boc)-OSu in IPAc was added at a rate such that the temperature of the reaction mixture remained below 30° C. The mixture was agitated for 1-2 hr. at approximately 20-30° C., or until reaction completion.

A quench solution was prepared by mixing sodium chloride (2.25 wt.), glacial acetic acid (0.15 wt) and water (9 vol.), and agitating the mixture until dissolution was achieved. One half of the quench solution was added to the reaction mixture, the mixture was agitated for 30-45 min., and the phases were separated. The remaining quench solution was added followed by agitation for 30-45 min. and phase separation.

The IPAc solution was washed with aqueous sodium bicarbonate solution (8% wt/wt, 11.5 vol.) and then brine (7% wt/wt, 11.5 vol.). The mixture was concentrated to 25-40% of its starting volume by vacuum distillation at ˜25-30° C. IPA (21 vol.) was added and the mixture was again concentrated to 25-40% of its starting volume by vacuum distillation at ˜25-30° C. IPA (2.5 vol.) was added, the mixture was filtered through a 0.45 mm filter, and rinsed with IPA (2.5 vol.).

Methane sulfonic acid (2.05 eq.) and water (0.6 vol.) were added to the filtrate, and the mixture was heated to ˜60-70° C. for 8 hr. at a partial vacuum (˜550 mmHg). IPAc (˜12.1 eq.) was added to the reaction mixture while maintaining its temperature at 60-70° C. The reaction mixture was stirred for 1-2 hrs. at 60-70° C., and cooled to ˜20-25° C. at a rate of approximately 10° C./hr. The mixture was heated to 60-70° C., stirred for 2-3 hrs., and cooled to approximately 20-25° C. at a rate of approximately 10° C./hr. The slurry was stirred for 16-16.5 hrs at 20-25° C. The product was filtered, washed twice with a mixture of IPA and IPAc (2:1, 10.5 vol. per wash), and dried at ˜65±5° C. under full vacuum. The dried material was milled through a 16-20 mesh screen to yield the final crystalline product.

Optical micrographs of the crystals prepared are in FIGS. 73 and 74. SEM images of the crystals prepared are shown in FIGS. 75 and 76.

Particle sizes, in terms of d₁₀, d₅₀ and d₉₀ values, were determined to be 4.1 μm, 19.5 μm and 60.8 μm, respectively.

Example 40 Characterization of Crystalline L-lysine-d-amphetamine Dimesylate

XRPD

The XRPD spectra of a sample of the crystalline L-lysine-d-amphetamine dimesylate prepared according to example 39 is shown in FIG. 77. Peak locations (in degrees 2Θ±0.2, 0.1, 0.05, or 0.01° C.) for the XRPD pattern in FIG. 77 are provided in table 73, below.

TABLE 73 Peak locations 2Θ d space (Å) Intensity  4.5 ± 0.2 19.534 ± 0.441  100  9.0 ± 0.2 9.805 ± 0.110 19 12.0 ± 0.2 7.400 ± 0.062 24 15.7 ± 0.2 5.637 ± 0.036 33 16.3 ± 0.2 5.453 ± 0.034 6 17.2 ± 0.2 5.143 ± 0.030 11 17.5 ± 0.2 5.061 ± 0.029 3 17.8 ± 0.2 4.971 ± 0.028 9 18.1 ± 0.2 4.903 ± 0.027 13 18.5 ± 0.2 4.785 ± 0.026 4 18.9 ± 0.2 4.684 ± 0.025 29 19.2 ± 0.2 4.624 ± 0.024 18 19.8 ± 0.2 4.492 ± 0.023 8 20.5 ± 0.2 4.333 ± 0.021 12 20.8 ± 0.2 4.277 ± 0.020 6 21.6 ± 0.2 4.123 ± 0.019 3 22.8 ± 0.2 3.899 ± 0.017 31 23.3 ± 0.2 3.814 ± 0.016 11 23.5 ± 0.2 3.782 ± 0.016 5 23.9 ± 0.2 3.719 ± 0.015 14 24.1 ± 0.2 3.691 ± 0.015 25 24.6 ± 0.2 3.615 ± 0.015 7 25.2 ± 0.2 3.532 ± 0.014 3 25.5 ± 0.2 3.489 ± 0.013 7 26.5 ± 0.2 3.366 ± 0.013 2 27.3 ± 0.2 3.273 ± 0.012 9 27.8 ± 0.2 3.215 ± 0.011 8 28.9 ± 0.2 3.092 ± 0.011 2 29.7 ± 0.2 3.007 ± 0.010 5

DSC

DSC thermograms were generated for thirteen different samples of L-lysine-d-amphetamine dimesylate, which were prepared according to the procedure in example 38 or 39. Each sample exhibited three endothermic events at approximately 94° C., 170° C. and 194° C. A representative DSC thermogram is shown in FIG. 78.

FT-Raman Spectra

The FT-Raman spectra of L-lysine-d-amphetamine dimesylate prepared according to the procedure in example 38 or 39 are shown in FIGS. 79 and 80. This crystal form of L-lysine-d-amphetamine dimesylate exhibits characteristic peaks at one or more of 1003, 1042, 2918, 2935, and 3055 cm⁻¹. The peaks observed in the FT-Raman spectra and their relative intensities are provided below in tables 74 and 75.

TABLE 74 Peak list for the FT-Raman Spectrum in FIG. 79 Position Intensity Position Intensity 125 40.311  229 18.864 286 6.445  344 20.104 392 7.222  426 3.248 451 3.964  464 4.618 496 3.758  511 6.449 530 7.205  556 21.639 601 3.070  621 7.989 683 2.113  745 5.327 778 25.979  814 6.988 849 2.680  863 4.269 876 4.851  904 5.906 942 3.913  958 3.471 1003 44.089 1042 46.907 1085 8.540 1097 7.210 1158 9.285 1182 12.150 1208 26.723 1263 7.095 1273 6.033 1291 7.508 1304 7.078 1332 10.244 1342 12.031 1378 3.474 1420 10.636 1446 20.092 1500 3.111 1550 3.080 1585 12.398 1601 12.963 1607 17.523 1656 13.423 2656 2.107  273: 2.911 2785 2.876 2852 7.447 2872 10.363 2918 46.220 2935 81.701  2978: 24.925 3004 16.222  3017: 19.421 3042 14.820 3055 33.276 3319 1.417

TABLE 75 Peak list for the FT-Raman Spectrum in FIG. 80 Position Intensity Position Intensity 125 40.311 229 18.864 344 20.104 392 7.222 556 21.639 621 7.989 778 25.979 814 6.988 876 4.851 904 5.906 1003 44.089 1042 46.907 1085 8.540 1182 12.150 1208 26.723 1342 12.031 1446 20.092 1585 12.398 1607 17.523 1656 13.423 2918 46.220 2935 81.701 2978 24.925 3017 19.421 3055 33.276

Single Crystal Analysis

A single crystal analysis was performed as follows. The l-lysine-d-amphetamine dimesylate was re-crystallized with a 1:1 acetonitrile/ethanol mixture and slurried at elevated temperature in acetonitrile overnight to obtain crystals suitable single crystal X-ray structure determination.

Single crystal data for the L-lysine-d-amphetamine dimesylate crystals was determined with Mo K_(α) radiation (λ=0.71073 Å) on a Nonius KappaCCD diffractometer equipped with a graphite crystal, incident beam monochromator. Refinements were performed on an LINUX PC using SHELX97.

Cell constants and an orientation matrix for data collection were obtained from least-squares refinement using the setting angles of 13580 reflections in the range 2°<θ<24°. The space group was determined by the program XPREP. From the systematic presence of the following conditions: 0k0 k; =2n, and from subsequent least-squares refinement, the space group was determined to be P 2₁ (no. 4). This is a chiral space group.

The data were collected to a maximum 2θ value of 48.23°, at a temperature of 150±1 K.

Frames were integrated with DENZO-SMN. A total of 13580 reflections were collected, of which 5474 were unique. Lorentz and polarization corrections were applied to the data. The linear absorption coefficient is 0.270 mm⁻¹ for Mo K_(α) radiation. An empirical absorption correction using SCALEPACK was applied. Transmission coefficients ranged from 0.884 to 0.987. Intensities of equivalent reflections were averaged. The agreement factor for the averaging was 13% based on intensity.

The structure was solved by direct methods using SIR2004. The remaining atoms were located in succeeding difference Fourier syntheses. Hydrogen atoms were included in the refinement but restrained to ride on the atom to which they are bonded. The structure was refined in full-matrix least-squares by minimizing the function:

Σw(|F_(o)|²−|F_(c)|²)²

The weight w is defined as 1/[(σ²(F_(o) ²)+(0.1193P)²+(9.5997P)], where P=(F_(o) ²+2F_(c) ²)/3.

Scattering factors were taken from the “International Tables for Crystallography”. Of the 5474 reflections used in the refinements, only the reflections with F_(o) ²>2σ(F_(o) ²) were used in calculating R. A total of 5089 reflections were used in the calculation. The final cycle of refinement included 546 variable parameters and converged (largest parameter shift was essentially equal to its estimated standard deviation) with unweighted and weighted agreement factors of:

$\begin{matrix} {{R = {{\sum{{{F_{o} - F_{c}}}/{\sum\; F_{o}}}} = 0.091}}} \\ {{R_{w} = {\sqrt{\left( {\sum{{w\left( {F_{o}^{2} - F_{c}^{2}} \right)}^{2}/{\sum{w\left( F_{o}^{2} \right)}^{2}}}} \right)} = 0.228}}} \end{matrix}$

The standard deviation of an observation of unit weight was 1.119. The highest peak in the final difference Fourier had a height of 0.68 e/Å³. The minimum negative peak had a height of −0.47 e/Å³. The factor for the determination of the absolute structure refined to 0.2(2).

The results of this analysis are shown in table 76 below.

TABLE 76 Indexed and Single Crystal lattice Parameters for Lisdexamphetamine Dimesylate Single Crystal Data Indexed Solution Form/Pattern Single Crystal Solution Dimesylate Family and Monoclinic Monoclinic Space Group P2₁ #4 P2₁/a #14 Z′/Z 2/4 1/4 a (Å) 10.2509(6) 10.304 b (Å) 11.2804(6) 11.355 c (Å) 19.3534(12) 19.499 α (deg) 90 90 β (deg) 94.124(3) 93.65 γ (deg) 90 90 Volume (Å³/cell) 2232.1(2) 2277 V/Z (Å³/asym. unit) 558 569.3 Density (g/cm³)^(a) 1.359 1.329 Weight Fraction Solvent 0 0 (%)^(a)

ORTEP and Packing Diagrams

ORTEP diagrams were prepared using the ORTEP III program within the PLATON software package. These diagrams are shown in FIGS. 81 and 82. Atoms are represented by 50% probability anisotropic thermal ellipsoids.

Packing diagrams were prepared using CAMERON modeling software. These diagrams are shown in FIGS. 83-85. Assessment of chiral centers, void calculations and additional figures were performed with the PLATON software package. Absolute configuration was evaluated using the specification of molecular chirality rules. Hydrogen bonding is represented as dashed lines.

XRPD Comparison

A comparison of a calculated XRPD pattern from the single crystal parameters, and the XRPD pattern of lisdexamphetamine dimesylate prepared according to Example 38 is shown in FIG. 86.

Example 41 Time Onset and Duration of Efficacy of L-lysine-d-amphetamine Dimesylate in Pediatric Clinical Trials

A randomized, double-blind, multi-center, placebo-controlled, two-way crossover, analog classroom study with an open-label optimization phase, designed to assess the time of onset, duration of efficacy, tolerability and safety of l-lysine-d-amphetamine dimesylate (30, 50, and 70 mg) was performed. The subjects of the study were children (males and females) ages 6-12 years old (n=129) who satisfied DSM-IV-TR criteria for a primary diagnosis of ADHD, combined or hyperactive-impulse subtype. The study included four phases: (1) Screening and washout; (2) open-label dose optimization; (3) analog classroom sessions 1 and 2; and (4) 30-day safety follow-up. Subjects were required to visit the clinic up to 8 times over a 7-10 week period.

Subjects were also required to have a baseline ADHD Rating Scale IV (ADHD-RS-IV) score ≧28, age-appropriate intellectual functioning, ability to complete the Permanent Product Measure of Performance (PERMP) assessment, blood pressure measurements within the 95th percentile for age, gender, and height.

Key exclusion criteria included the presence of a comorbid psychiatric condition with severe symptoms, conduct disorder, or other medical condition that could, in the judgment of the physician investigator, confound efficacy or safety assessments, pose a risk to the subject, or prohibit study completion. Children who had an adverse reaction (allergy, hypersensitivity, intolerance) or who failed to respond to previous amphetamine therapy were not allowed to participate in the study. Other exclusion criteria were positive serum or urine pregnancy test, history of substance abuse, weight <22.7 kg (50 lb), BMI >98th percentile for age, seizure within the last 2 years, tic or Tourette disorder, use of medication with effects on CNS function or performance, and clinically significant laboratory and ECG abnormalities. Children whose current ADHD medication provided effective control of symptoms with acceptable tolerability were also excluded.

Screening and Washout

Subjects were screened for approximately 3-weeks to establish eligibility for study participation. Those subjects who met eligibility requirements underwent medication washout, if applicable. The length of the medication washout (if applicable) was no longer than 7 days depending upon the half-life of the subject's current ADHD medication.

Open-Label Dose Optimization

Following screening and washout, eligible subjects entered the open-label dose optimization phase, during which they received 1 capsule of l-lysine-d-amphetamine dimesylate in an openlabel manner and were evaluated for efficacy and tolerability to that dosage approximately 7 days later. Dosage was initiated at 30 mg/d l-lysine-d-amphetamine dimesylate and adjusted to the next available dose, until optimal dose was reached. Optimal dose was defined as the dose that produced a reduction in ADHD-RS-IV score ≧30% and CGI-I score of 1 or 2 and had tolerable side effects. Tolerability was determined by the investigator, based on review of AEs and clinical judgement. Once reached, the optimal dose was maintained for the remainder of the dose optimization phase and was used for the double-blind treatment sequence phase.

Dose increase was permitted if the current dose was well tolerated, produced a reduction in ADHD-RS-IV score ≧30% and CGI-I score of 1 or 2, and if the next available dose would, in the opinion of the clinician, provide additional symptom reduction. Dose reduction was permitted if tolerability to the current dose was unacceptable; only 1 dose reduction was allowed. Subjects were discontinued if they were unable to tolerate l-lysine-d-amphetamine dimesylate or had not reached their optimal dose by visit 4/week 4. The dose dispensed at visit 3 was used at visit 4 and the during the double-blind treatment sequence period. During visit 4, a practice analog classroom session was conducted, to familiarize subjects with classroom schedules and procedures. Three to 5 practice PERMP tests were also given during the practice session.

Double Blind Crossover Treatment with Analog Classroom Sessions 1 and 2

Following completion of the open-label dose optimization period and successful titration to an optimal dose of l-lysine-d-amphetamine dimesylate, subjects entered the 2 week double blind treatment portion of the study. Subjects were randomized in a 1:1 ratio to 1 of 2 treatment sequences: daily l-lysine-d-amphetamine dimesylate treatment (at the optimized dose) for one week followed by daily placebo treatment (administered as matched capsules identical in appearance to l-lysine-d-amphetamine dimesylate capsules) treatment for one week, or vice versa.

For the first 6 days of each week during the double-blind treatment phase, the study drug was administered by the parent. On the last day of each week, the daily dose was administered by study staff in the analog classroom, where efficacy and safety assessments were also performed.

Each analog classroom had 10 to 18 subjects, with a maximum of 16 from the same cohort. Subjects arrived at 6 AM, were administered the SKAMP and PERMP at 0.5 hours predose (6:30 AM), and received their randomized treatment (7 AM). SKAMP and PERMP were then administered in the analog classroom at 1.5, 2.5, 5, 7.5, 10, 12 and 13 hours postdose. Subjects then departed at approximately 8:30 PM.

Primary Efficacy

Efficacy was measured by the SKAMP score (teacher rating scale) to evaluate the behavioral effects of l-lysine-d-amphetamine dimesylate compared to placebo, under controlled conditions, measured at multiple time points throughout the day. The SKAMP was designed for independent observers to rate 13 items representing two factors of classroom behavior—attention and deportment. A detailed description of SKAMP can be found elsewhere (Wigal S B, Gupta S, Guinta D, Swanson J M. Reliability and validity of the SKAMP rating scale in a laboratory school setting. Psychopharmacol Bull. 1998; 34:47-53).

In this study, a single, independent, trained observer rated each subject on 13 items, using a 7-point impairment scale (0=normal, 6=maximal impairment). Onset and duration of efficacy were determined through SKAMP deportment assessments at 0.5 hours predose and 1.5, 2.5, 5, 7.5, 10, 12 and 13 hours postdose.

The primary efficacy measure was the onset of action measured by the mean SKAMP-DS score. A mixed linear model was used to analyze the mean SKAMP-DS score as well as the SKAMP-DS scores for each time point. In this model, the fixed effects were sequence, period, and treatment, while the random effect was subject-within-sequence. Raw means and effect sizes, least-squares (LS) means and effect sizes, differences in LS means and 95% confidence interval for the difference between treatment groups, p values, and model result were calculated for each postdose timepoint and for mean scores for the intent to treat (ITT) population, i.e., the population which consisted of all randomized subjects with at least 1 postrandomization measurement of the primary efficacy variable (mean SKAMP deportment score over the course of a day) available for analysis.

Secondary Efficacy

Secondary efficacy measures included the SKAMP subscales of attention, quality of work, and compliance, SKAMP total score, PERMP-A and -C, ADHD-RS-IV and the Clinical Global Impressions (CGI). The PERMP is a 5-page math test consisting of 80 problems each (total of 400 problems) and was used to evaluate efficacy and to determine time of onset. Subjects were instructed to work at their desks and to complete as many problems as possible in 10 minutes. Academic performance was evaluated using two scores: PERMP-A (the number of problems attempted) and PERMP-C (the number of problems correct). The appropriate level of difficulty for each student was determined based on results of a math pre-test administered at screening. The PERMP was completed during analog classroom sessions at the same time points as the SKAMP scale. To avoid taking the same test more than once during the study, subjects were given a different version of the test at each assessment time point.

The clinician completed ADHD-RS-IV was also used as a secondary efficacy measure. The ADHD-RS-IV was administered at each visit, beginning with Screening, and at baseline and each visit, to capture the ADHD symptoms within each study week. The ADHD-RS-IV consists of 18 items designed to reflect current symptomatology of ADHD based on DSM-IV-TR™ criteria. The 18 items are grouped into 2 subscales (hyperactivity/impulsivity and inattentiveness). Each item is scored on a scale of 0 (no symptoms) to 3 (severe symptoms), yielding a total score of 0 to 54.

In addition to the ADHD-RS-IV, the Clinical Global Impressions (CGI) Scale was used as a secondary efficacy measure. The CGI rating scale permits a global evaluation of the subject's improvement over time. At Baseline, a CGI-S assessment was performed, in which the Investigator rates the severity of a subject's condition on an 8-point scale ranging from 0 (not assessed) to 7 (among the most extremely ill).

Responses for the CGI-I were dichotomized such that “very much improved” (CGI-I score of 1) and “much improved” (CGI-I score of 2) were combined into one category (“improved”), and the remaining responses were combined into the other category (“not improved”).

The mixed linear model used to analyze scores for the SKAMP-DS onset of action was also used to analyze scores for the secondary efficacy measures.

Statistical Analysis

Determination of sample size was based on analysis of SKAMP deportment scores from a previous crossover study scale 10. Assuming a standard deviation SD) of 0.9491 (the maximum SD reported in the previous crossover study), 96 subjects (48 subjects in each treatment sequence) would need to complete the study to detect a difference of 0.50 in mean SKAMP deportment scores between placebo and l-lysine-d-amphetamine dimesylate at 95% power (P=0.05, 2-sided). However, 128 subjects were planned for enrollment, since 25% of subjects were predicted to discontinue during the study. All statistical tests were 2-sided and performed at the 0.05 significance level.

Efficacy Outcomes.

As stated above, efficacy was demonstrated at each post-dose timepoint assessed, starting at 1.5 hrs. postdose, and continuing through the last (13.0 hrs. postdose). The primary efficacy results are given in FIG. 88. At all timepoints, L-lysine-d-amphetamine dimesylate had significantly lower LS mean SKAMP deportment scores (DS) compared with placebo (P≦0.01).

Secondary efficacy measurements were also demonstrated at each postdose timepoint. As with the primary measurements, L-lysine-d-amphetamine dimesylate had significantly lower LS mean SKAMP attention scores compared with placebo, at all timepoints (P<0.0001). These results are given in FIG. 89.

The results from the PERMP-A tests are given in FIG. 90. Efficacy was demonstrated at each post-dose timepoint assessed, starting at 1.5 hrs. postdose, and continuing through the last (13.0 hrs. postdose). At all timepoints, L-lysine-d-amphetamine dimesylate had significantly higher PERMP-A scores compared with placebo (P≦0.0001).

The results from the PERMP-C tests are given in FIG. 91. Efficacy was demonstrated at each post-dose timepoint assessed, starting at 1.5 hrs. postdose, and continuing through the last (13.0 hrs. postdose). At all timepoints, L-lysine-d-amphetamine dimesylate had significantly higher PERMP-A scores compared with placebo (P≦0.0001). As FIGS. 88-91 indicate, L-lysine-d-amphetamine dimesylate has a duration of action (as measured by efficacy) for at least 13 hours.

An analysis of the change from Baseline in ADHD-RS-IV Total scores, Inattention subscale scores, and Hyperactivity/Impulsivity subscale scores for the Cross-Over phase (Visit 5/6) is presented for the ITT population in table 77 below. For all three ADHD-RS-IV measures, l-lysine-d-amphetamine dimesylate treatment resulted in a larger reduction from Baseline scores compared to placebo treatment. For all three ADHD-RS-IV measures, change from Baseline scores associated with l-lysine-d-amphetamine dimesylate treatment were statistically significantly different (p<0.0001) from those associated with placebo treatment.

TABLE 77 Analysis ADHD-RS-IV Change from Baseline Total and Subscale Scores at Visit 5/6 by Treatment (ITT population) Raw LS effect efect Diff. Raw mean (SE) size LS mean (SE) size in LS 95% variable Drug placebo (SE) Drug placebo (SE) means C.L. P value total score −25.78 −8.65 −1.33 −25.75 −8.66 −1.36 −17.1 −20.41, <0.0001 (1.22) (1.20) (0.16) (1.20) (1.20) (0.16) −13.78 Inattention score 12.47 −4.12 −1.24 −12.45 −4.12 −1.35 −8.33 −9.96, <0.0001 (0.65) (0.61) (0.16) (0.62) (0.62) (0.16) −6.71 Hyperactivity/impulsivity 13.31 −4.53 −1.28 13.30 −4.54 −1.31 −8.76 −10.52, <0.0001 score (0.64) (0.65) (0.16) (0.64) (0.64) (0.16) −7.00

An analysis of CGI-I scores for the Cross-Over Phase (Visits 5 and 6) is presented in graphically in FIG. 92. A majority of the 113 subjects in the ITT population (71.7%) were improved while receiving l-lysine-d-amphetamine dimesylate but not on placebo. Ten subjects (8.8%) were not improved while receiving l-lysine-d-amphetamine dimesylate but were improved on placebo. The difference between l-lysine-d-amphetamine dimesylate and placebo treatment was statistically significant (p<0.0001).

Example 42 Within and Between Subject Variability for Patients Administered l-lysine-d-amphetamine Dimesylate

An open-label, five-period, five-treatment, dose escalating study of the safety, tolerability, and pharmacokinetics of l-lysine-d-amphetamine dimesylate administered as a single oral dose was conducted. The study was conducted at a single center and consisted of a Screening and five single Dosing Periods. During the Screening Period (within 28 days prior to Dosing Period 1, Day 1 dosing), the eligibility of subjects were confirmed. Eligibility was re-confirmed at check-in on Day 1. Eligible participants were confined to the Clinical Research Center (CRC) after checking in on Day 1 and remained in the CRC until approximately 96 hours post Dosing Period 5 and after the scheduled discharge assessments and procedures were completed. No washout period was given between dosing periods. The total duration of the confinement for this study was 22 days.

Subjects received a single dose of l-lysine-d-amphetamine dimesylate on Day 1 of each Dosing Period and a total of five doses over the course of the study. During each Dosing Period, serial blood samples were collected up to 96 hours post dose. During this time, subjects were required to fast from approximately 10:00 pm the night before receiving study drug until four hours after taking study drug. Subjects were discharged from the CRC at least 96 hours after taking the last dose of study medication in Dosing Period 5 and once all discharge assessments and procedures were complete and unremarkable. Approximately 7 days after the last dose of study drug was administered, a follow-up was done to report any ongoing concomitant medication(s), adverse events (AEs), and/or new AEs or Serious Adverse Events (SAEs).

Pharmacokinetic Analysis:

Descriptive statistics (number of observations [N], mean, standard deviation, coefficient of variation [CV], median, minimum, and maximum) were used to summarize plasma concentration at each planned sampling time point for each treatment. Plasma pharmacokinetic parameters calculated from the concentrations were also summarized by dose level using descriptive statistics.

Dose proportionality of the key pharmacokinetic parameter AUC_(0-∞) over the administered dose range for d-amphetamine was based on the following power model:

log(parameter)=a+b*log(dose); where ‘a’ is the intercept and ‘b’ is the slope.

The power model fitted for the log-transformed parameter AUC_(0-∞) with a mixed model included a random intercept. The estimated mean slope (‘b’) and 90% Confidence Intervals (CIs) were constructed. Linearity was assessed based on a test of the null hypothesis that the slope is equal to 1 versus the alternative hypothesis that the slope is not equal to 1. The other pharmacokinetic parameters were summarized as described above.

Pharmacokinetic Evaluations

On Day 1 of each dosing period, blood samples for pharmacokinetic analysis were drawn pre-dose and, after a single oral dose of l-lysine-d-amphetamine dimesylate, at 0.25, 0.5, 1, 1.5, 2, 4, 6, 8, 12, 16, 24, 36, 48, 72 and 96 hours post-dose. On Day 5, after collecting the 96-hour post dose sample, subjects progressed to the next dosing period. The 96-hour sample served as the pre-dose sample for the next period. All pharmacokinetic data are presented by dose (50, 100, 150, 200, 250 mg).

The mean d-amphetamine plasma concentration profiles for all five lisdexamfetamine dimesylate dosing periods (50, 100, 150, 200, 250 mg) are shown in FIG. 93. Other pharmacokinetic parameters measured in this study are given in table 78 below. Across all doses administered, pharmacokinetic parameters were consistent and well-behaved. The mean maximum observed concentration (C_(max)) and mean systemic exposure (AUC_(0-t), AUC_(0-∞)) increased proportionately with dose. The mean time to maximum plasma concentration (t_(max)) occurred at approximately 5 hours and the mean terminal half life (t_(1/2)) at approximately 11.5 hours.

TABLE 78 Mean Plasma Pharmacokinetic Parameters by Dose for d-amphetamine after a Single Oral Dose of Lisdexamfetamine Dimesylate Lisdexamfetamine Dimesylate Dose 50 mg 100 mg 150 mg 200 mg 250 mg C_(max) (ng/mL) N 20 20   18 12   9 Mean 44.62 84.55 126.57 168.83  246.32 (SD) (9.31)   15.07) 29.47)   50.63) 100.81) % CV 20.9 17.8  23.3 30   40.9 Median 43 84.79 117.61 175.77  230.49 (Min, Max) (33.58, 61.14)   (66.96, 115.78)  (89.75, 190.07) (43.26, 234.53) (148.2, 477.19) t_(max), hr N 20 20   18 12   9 Mean 4 4.5 4.9 5.7 5.8 (SD) (1.2) (0.9) (1.6) (0.8) (1.2) % CV 29.6 19.7  32 13.8  20.8 Median 4 4   4 6   6 (Min, Max) (1.5, 6)   (4, 6) (2, 8)  (4, 6.1) (4, 8)   AUC_(0-t) (ng · h/mL)^(a) N 20 20   18 12   9 Mean 763.1 1485.1   2429.3 3265.5   5056.8 (SD) (190.4) (401.2)  (727.2) (1207.7)   (1419.8) % CV 24.9 27   29.9 37   28.1 Median 732 1413.9   2237.9 3359.9   4924.3 (Min, Max) (546.8, 1382.9) (1105.9, 959.1)  (1511.8, 792.4)  (727.8, 5945.9)  (3173, 8394.2) AUC_(0-∞) (ng · h/mL) N 20 20   18 12   9 Mean 818.1 1548.2   2503.4 3336.2   5132.5 (SD) (194.6) (396.3)  (723.3) (1212.7)   (1464.5) % CV 23.8 25.6  28.9 36.3  28.5 Median 793.5 1468.2   2318 3482    5006.4 (Min, Max) (622.2, 1446.7) (1166.6, 3004.3) (1551.7, 4842.7) (774.5, 6014.7) (3232.7, 8605)   t_(1/2), hr N 20 20   18 12   9 Mean 11.3 11.1  10.9 11.3  12.4 (SD) (2.4) (2)   (2.1) (2)   (2.3) % CV 21 18.5  19.6 17.5  18.9 Median 10.8 10.6  10.6 11.6  11.7 (Min, Max) (7.6, 16)     (8, 15.1 )  (8.3, 15.7) (7.8, 14.1 ) (10.4, 17.9)  ^(a)AUC_(0-t) is defined as the area under the plasma concentration-time curve from time zero to the last sampling time at which plasma concentrations were measurable

Dose Proportionality

Systemic exposure (AUC_(0-∞)) to d-amphetamine increased in a dose proportional manner over the range of 50-250 mg. Analysis of the linearity (table 79) of the AUC_(0-∞) vs. dose curve yielded regression parameters that confirm dose proportionality. The slope deviated slightly from linearity due to the presence of outliers at the two higher doses.

TABLE 79 Dose Proportionality of plasma d-amphetamine AUC_(0-∞) after Oral Administration of l-lysine-d-amphetamine AUC_(0-∞) (hr * ng/mL) Regression^(a) Slope (SE) 1.1 (0.058) (90% CI, ln scale) (0.962, 1.16) ^(a)Assessment of dose proportionality based on the power model: log (ln AUC_(0-∞)) = a + b * log (dose); where “a” is the intercept and “b” is the slope.

The maximum observed plasma d-amphetamine concentration (C_(max)) was also dose proportional over the dose range of 50-250 mg. Analysis of the linearity of the C_(max) vs. dose curve (Table 80) yielded regression parameters that confirmed a linear relationship of C_(max) with dose.

TABLE 80 Dose Proportionality of plasma d-amphetamine for C_(max) after Oral Administration of Lisamfetamine Dimesylate C_(max) (ng/mL) Regression^(a) Slope (SE) 1.0 (0.054) (90% CI, ln scale) (0.891, 1.07) ^(a)Assessment of dose proportionality based on the power model: log (ln C_(max)) = a + b* log (dose); where “a” is the intercept and “b” is the slope.

Intra- and Inter-Subject Variability

Within-subject (intra-subject) and between-subject (inter-subject, or patient to patient) variability in d-amphetamine AUC_(0-∞) and C_(max) are presented for all doses (Table 81); and for only the 50, 100 and 150 mg dose groups (Table 82) are presented in tables 81 and 82, below.

For AUC_(0-∞), when data from all dosing periods was considered, total variability was about 28%. Within-subject and between subject variability were similar (about 20%). When data from the three lowest (50, 100, 150 mg) dosing periods was considered (Table 82), total variability in AUC_(0-∞) was reduced (22%) and the majority of the variability was between-subject. The within-subject variability was about half that of the between-subject variability (10% vs. 20%). For C_(max), when data from all dosing periods was considered, total variability was about 27%. Between-subject was less than within-subject variability (16% vs. 22%). When data from the three lowest (50, 100, 150 mg) dosing periods was considered, total variability in C_(max) was reduced (20%) and the majority of the variability was between-subject. The within-subject variability was about half that of the between-subject variability (9% vs. 18%).

TABLE 81 Within- and between-subject variability for d-amphetamine - all lysine-d- amphetamine dimesylate dimesylate doses Within-Subject Between-Subject Total Parameter Estimate 95% CI Estimate 95% CI Estimate 95% CI Log AUC_(0-∞) 0.195 (0.164, 0.240) 0.204 (0.139, 0.316) 0.282 (0.238, 0.374) Log C_(max) 0.215 (0.181, 0.264) 0.163 (0.096, 0.264) 0.269 (0.232, 0.346) Note: Variability is on the SD (standard deviation) scale for log-transformed parameters.

TABLE 82 Within- and between-subject variability for d-amphetamine - 50, 100 and 150 mg - lysine-d-amphetamine dimesylate dimesylate doses Within-Subject Between-Subject Total Parameter Estimate 95% CI Estimate 95% CI Estimate 95% CI Log AUC_(0-∞) 0.104 (0.085, 0.135) 0.197 (0.143, 0.294) 0.222 (0.177, 0.313) Log C_(max) 0.092 (0.074, 0.119) 0.176 (0.129, 0.263) 0.198 (0.158, 0.279) Note: Variability is on the SD (standard deviation) scale for log-transformed parameters.

Pharmacokinetic Conclusions

Plasma d-amphetamine vs. time curves for doses from 50-250 mg were similar in shape and demonstrated well-behaved, consistent, dose-proportional behavior.

Pharmacokinetic parameters for d-amphetamine were also dose-proportional (AUC_(0-∞), C_(max)) consistent (t_(max), t_(1/2)), and well-behaved over the whole dose range of 50 mg-250 mg. In this study, systemic exposure, maximum plasma concentration, time to maximum concentration and the elimination half life were reliably predictable even at supratherapeutic doses (more than 3.5 times the current maximum daily dose).

Intra-subject variability in d-amphetamine AUC_(0-∞) and C_(max) was low, particularly when the three lowest doses (50-150 mg) were evaluated. For AUC_(0-∞), in the 50-150 mg dose group, the majority of the total variability (22%) arose from between-subject variation (20%). Intra-subject variability (10%) was approximately half the inter-subject variability.

It will be understood that the specific embodiments of the invention shown and described herein are exemplary only. Numerous variations, changes, substitutions and equivalents will occur to those skilled in the art without departing from the spirit and scope of the invention. In particular, the terms used in this application should be read broadly in light of similar terms used in the related applications. Accordingly, it is intended that all subject matter described herein and shown in the accompanying drawings be regarded as illustrative only and not in a limiting sense and that the scope of the invention be solely determined by the appended claims. 

1. Crystalline lisdexamphetamine dimesylate.
 2. The crystalline lisdexamphetamine dimesylate of claim 1, wherein the crystalline lisdexamphetamine dimesylate exhibits an X-ray powder diffraction pattern substantially as shown in FIG.
 77. 3. The crystalline lisdexamphetamine dimesylate of claim 1, wherein the crystalline lisdexamphetamine dimesylate exhibits an X-ray powder diffraction pattern having at least one peak in degrees 2ÿ±0.2 2ÿ selected from 4.5, 9.0, 12.0, 15.7, and 16.3.
 4. The crystalline lisdexamphetamine dimesylate of claim 3, wherein the dimesylate exhibits an X-ray powder diffraction pattern having at least two peaks in degrees 2Θ±0.2 2Θ selected from 4.5, 9.0, 12.0, 15.7, and 16.3.
 5. The crystalline lisdexamphetamine dimesylate of claim 4, wherein the dimesylate exhibits an X-ray powder diffraction pattern having peaks in degrees 2Θ±0.2 2Θ at 4.5, 9.0, 12.0 and 15.7.
 6. Crystalline lisdexamphetamine dimesylate exhibiting an X-ray powder diffraction pattern having at least one peak in degrees 2Θ±0.2 2Θ selected from 4.5, 9.0, 12.0, 15.7, and 16.3.
 7. The crystalline lisdexamphetamine dimesylate of claim 1, wherein the crystalline lisdexamphetamine dimesylate has a melting point onset as determined by differential scanning calorimetry at about 194.7° C.
 8. The crystalline lisdexamphetamine dimesylate of claim 1, wherein the crystalline lisdexamphetamine dimesylate exhibits a single crystal X-ray crystallographic analysis at 150 K with crystal parameters that are approximately equal to the following: Parameter Value Space group P2₁/a Cell Dimensions a (Å) 10.25 b (Å) 11.28 c (Å) 19.35 ( ) 94.12 Volume (Å³) 2232.1 Z (Molecules/unit cell) 4 Density (g/cm³) 1.359


9. Crystalline lisdexamphetamine dimesylate particles having at least one of: (a) a d₁₀ ranging from about 1 to about 10 μm; (b) a d₅₀ ranging from about 8 to about 40 μm; and (c) a d₉₀ particle size ranging from about 25 to about 90 μm.
 10. The crystalline lisdexamphetamine dimesylate particles of claim 9, wherein the crystalline lisdexamphetamine dimesylate has a d₉₀ particle size ranging from about 25 to about 90 μm.
 11. The crystalline lisdexamphetamine dimesylate particles of claim 9, wherein the crystalline lisdexamphetamine dimesylate has a d₅₀ ranging from about 8 to about 40 μm.
 12. (canceled)
 13. A pharmaceutical composition comprising crystalline lisdexamphetamine dimesylate of any one of claim 1 and a pharmaceutically acceptable additive.
 14. The pharmaceutical composition of claim 13, wherein the pharmaceutical composition comprises at least about 95% by weight of crystalline lisdexamphetamine dimesylate, based upon 100% total weight of lisdexamphetamine dimesylate in the pharmaceutical composition.
 15. A method of treating attention deficit hyperactivity disorder in a patient in need thereof, comprising the step of orally administering the crystalline lisdexamphetamine dimesylate of claim
 1. 16.-67. (canceled) 